Group b streptococcus polypeptides nucleic acids and therapeutic compositions and vaccines thereof

ABSTRACT

This invention provides isolated nucleic acids encoding polypeptides comprising amino acid sequences of streptococcal matrix adhesion (Ema) polypeptides. The invention provides nucleic acids encoding Group B streptococcal Ema polypeptides EmaA, EmaB, EmaC, EmaD and EmaE. The present invention provides isolated polypeptides comprising amino acid sequences of Group B streptococcal polypeptides EmaA, EmaB, EmaC, EmaD and EmaE, including analogs, variants, mutants, derivatives and fragments thereof. Ema homologous polypeptides from additional bacterial species, including  S. pneumoniae, S. pyogenes, E. faecalis  and  C. diptheriae  are also provided. Antibodies to the Ema polypeptides and immunogenic fragments thereof are also provided. The present invention relates to the identification and prevention of infections by virulent forms of streptococci. This invention provides pharmaceutical compositions, immunogenic compositions, vaccines, and diagnostic and therapeutic methods of use of the isolated polypeptides, antibodies thereto, and nucleic acids. Assays for compounds which modulate the polypeptides of the present invention for use in therapy are also provided.

GOVERNMENTAL SUPPORT

[0001] The research leading to the present invention was supported, atleast in part, by a grant from NAID, Grant No.A140918. Accordingly, theGovernment may have certain rights in the invention.

FIELD OF THE INVENTION

[0002] This invention relates generally to extracellular matrix adhesin(Ema) proteins, antibodies thereto and to vaccines, compositions andtherapeutics. The Group B streptococcal Ema polypeptides are EmaA, EmaB,EmaC, EmaD and EmaE. The invention further relates to Ema polypeptidesfrom various species of bacteria, including S. pneummoniae, S. pyogenes,E. faecalis and C. diptheriae. The invention also relates to theidentification and prevention of infections by streptococci. Isolatednucleic acids encoding Group B streptococcal Ema polypeptides,particularly EmaA, EmaB, EmaC, EmaD and EmaE and to other bacterial Emahomologs are included herein. Assays for compounds which modulate thepolypeptides of the present invention for use in therapy are alsoprovided.

BACKGROUND OF THE INVENTION

[0003] Streptococci are catalase negative gram positive cocci. They maybe classified by the type of hemolysis exhibited on blood agar, by theserologic detection of carbohydrate antigens, or by certain biochemicalreactions. Medically important streptococci include Groups A, B, D, S.pneumoniae and the viridans group of streptococci. Lancefield type A(GroupA) Streptococcus pyogenes is an important human pathogen—the causeof streptococcal pharyngitis, impetigo and more severe infections suchas bacteremia and necrotizing fascitis. The immunologic sequelae ofGroup A Streptococcal infections are also important healthproblems—rheumatic carditis is the most common cause of acquired cardiacdisease worldwide and post-streptococcal glomerulonephritis is a causeof hypertension and renal dysfunction. Group B Streptococcus agalactiaeare the most common cause of serious bacterial infections in newborns,and important pathogens in pregnant women and nonpregnant adults withunderlying medical problems such as diabetes and cardiovascular disease.Group D streptococci include the enterococci (Streptococcus faecalis andfaecium) and the “nonenterococcal” Group D streptococci. Streptococcuspneumoniae (pneumococcus) is not classified by group in the Lancefieldsystem. Pneumococci are extremely important human pathogens, the mostcommon cause of bacterial pneumonia, middle ear infections andmeningitis beyond the newborn period. The viridans group of streptococciinclude S. milleri, S. mitis, S. sanguis and others. They causebacteremia, endocarditis, and dental infections. Enterococci areimportant causes of urinary tract infections, bacteremia and woundinfections (predominantly as nosocomial infections in hospitalizedpatients), and endocarditis. Over the past decade enterococci havedeveloped resistance to many conventional antibiotics and there are somestrains resistant to all known antibiotics.

[0004] Group B streptococci (GBS) are the most common cause of seriousbacterial disease in neonates, and are important pathogens in pregnantwomen and adults with underlying illnesses (Baker C J. (2000) “Group Bstreptococcal infections” in Streptococcal infections. Clinical aspects,microbiology, and molecular pathogenesis. (D. L. Stevens and E. L.Kaplan), New York: Oxford University Press, 222-237). Commonmanifestations of these infections include bacteremia, pneumonia,meningitis, endocarditis, and osteoarticular infections (Baker C J.(2000) “Group B streptococcal infections” in Streptococcal infections.Clinical aspects, microbiology, and molecular pathogenesis. (D. L.Stevens and E. L. Kaplan), New York: Oxford University Press, 222-237;Blumberg H. M. et al. (1996) J Infect Dis 173:365-373). The incidence ofinvasive GBS disease is approximately 2.6 in 1000 live births and 7.7 in100,000 in the overall population, with mortality rates that vary from 6to 30% (Baker C J. (2000) “Group B streptococcal infections” inStreptococcal infections. Clinical aspects, microbiology, and molecularpathogenesis. (D. L. Stevens and E. L. Kaplan), New York: OxfordUniversity Press, 222-237; Blumberg H. M. et al. (1996) J Infect Dis173:365-373). Although much neonatal disease is preventable byadministration of prophylactic antibiotics to women in labor, antibioticprophylaxis programs can be inefficient, suffer from poor compliance, orfail if antibiotic resistance emerges. No effective prophylaxis strategyfor adult infections has been established.

[0005] During childbirth, GBS can pass from the mother to the newborn.By one estimate, up to 30% of pregnant women carry GBS at leasttemporarily in the vagina or rectum without symptoms. Infants born tothese women become colonized with GBS during delivery (Baker, C. J. andEdwards, M. S. (1995) “Group B Streptococcal Infections” in InfectiousDisease of the Fetus and Newborn Infant (J. S. Remington and J. OKlein), 980-1054). Aspiration of infected amniotic fluid or vaginalsecretions allow GBS to gain access to the lungs. Adhesion to, andinvasion of, respiratory epithelium and endothelium appear to becritical factors in early onset neonatal infection. (Baker, C. J. andEdwards, M. S. (1995) “Group B Streptococcal Infections” in InfectiousDisease of the Fetus and Newborn Infant (J. S. Remington and J. O.Klein), 980-1054; Rubens, C. E. et al. (1991) J Inf Dis 164:320-330).Subsequent steps in infection, such as blood stream invasion and theestablishment of metastatic local infections have not been clarified.The pathogenesis of neonatal infection occurring after the first week oflife is also not well understood. Gastrointestinal colonization may bemore important than a respiratory focus in late onset neonatal disease(Baker, C. J. and Edwards, M. S. (1995) “Group B StreptococcalInfections” in Infectious Disease of the Fetus and Newborn Infant (J. S.Remington and J. O Klein), 980-1054). Considerable evidence suggeststhat invasion of brain microvascular endothelial cells by GBS is theinitial step in the pathogenesis of meningitis. GBS are able to invadehuman brain microvascular endothelial cells and type III GBS, which areresponsible for the majority of meningitis, accomplish this 2-6 timesmore efficiently than other serotypes (Nizet, V. et al. (1997) InfectImmun 65:5074-5081).

[0006] Because GBS is widely distributed among the population and is animportant pathogen in newborns, pregnant women are commonly tested forGBS at 35-37 weeks of pregnancy. Much of GBS neonatal disease ispreventable by administration of prophylactic antibiotics during laborto women who test positive or display known risk factors. However, theseantibiotics programs do not prevent all GBS disease. The programs aredeficient for a number of reasons. First, the programs can beinefficient. Second, it is difficult to ensure that all healthcareproviders and patients comply with the testing and treatment. Andfinally, if new serotypes or antibiotic resistance emerges, theantibiotic programs may fail altogether. Currently available tests forGBS are inefficient. These tests may provide false negatives.Furthermore, the tests are not specific to virulent strains of GBS.Thus, antibiotic treatment may be given unnecessarily and add to theproblem of antibiotic resistance. Although a vaccine would beadvantageous, none are yet commercially available.

[0007] Traditionally, GBS are divided into 9 serotypes according to theimmunologic reactivity of the polysaccharide capsule (Baker C J. (2000)“Group B streptococcal infections” in Streptococcal infections. Clinicalaspects, nzicrobiology, and molecular pathogenesis. (D. L. Stevens andE. L. Kaplan), New York: Oxford University Press, 222-237; Blumberg H.M. et al. (1996) J Infect Dis 173:365-373; Kogan, G. et al. (1996) JBiol Chem 271:8786-8790). Serotype III GBS are particularly important inhuman neonates, causing 60-70% of all infections and almost allmeningitis (Baker C J. (2000) “Group B streptococcal infections” inStreptococcal infections. Clinical aspects, microbiology, and molecularpathogenesis. (D. L. Stevens and E. L. Kaplan), New York: OxfordUniversity Press, 222-237). Type III GBS can be subdivided into threegroups of related strains based on the analysis of restriction digestpatterns (RDPs) produced by digestion of chromosomal DNA with Hind IIIand Sse83 87 (I. Y. Nagano et al. (1991) J Med Micro 35:297-303; S.Takahashi et al. (1998) J Inf Dis 177:1116-1119).

[0008] Over 90% of invasive type III GBS neonatal disease in Tokyo,Japan and in Salt Lake City, Utah is caused by bacteria from one ofthree RDP types, termed RDP type III-3, while RDP type III-2 aresignificantly more likely to be isolated from vagina than from blood orCSF. These results suggest that this genetically-related cluster of typeIII-3 GBS are more virulent than III-2 strains and could be responsiblefor the majority of invasive type III disease globally.

[0009] Preliminary vaccines for GBS used unconjuated purifiedpolysaccaride. GBS poly- and oligosaccharides are poorly immunogenic andfail to elicit significant memory and booster responses. Baker et atimmunized 40 pregnant women with purified serotype III capsularpolysaccharide (Baker, C. J. et at. (1998) New Engl J of Med319:1180-1185). Overall, only 57% of women with low levels of specificantibody responded to the vaccine. The poor immunogenicity of purifiedpolysaccharide antigen was further demonstrated in a study in whichthirty adult volunteers were immunized with a tetravalent vaccinecomposed of purified polysaccharide from serotypes Ia, Ib, II, and III(Kotloff, K. L. et al. (1996) Vaccine 14:446-450). Although safe, thisvaccine was only modestly immunogenic, with only 13% of subjectsresponding to type Ib, 17% to type II, 33% responding to type Ia, and70% responding to type III polysaccharide. The poor immunogenicity ofpolysaccharide antigens prompted efforts to develop polysaccharideconjugate vaccines, whereby these poly- or oligosaccharides areconjugated to protein carriers. Ninety percent of healthy adult womenimmunized with a type III polysaccharide-tetanus toxoid conjugatevaccine responded with a 4-fold rise in antibody concentration, comparedto 50% immunized with plain polysaccharide (Kasper, D. L. et at (1996) Jof Clin Invest 98:2308-2314). A type Ia/Ib polysaccharide-tetanus toxoidconjugate vaccine was similarly more immunogenic in healthy adults thanplain polysaccharide (Baker, C. J. et at (1999) J Infect Dis179:142-150).

[0010] The disadvantage of polysaccharide-protein conjugate vaccines isthat the process of purifying and conjugating polysaccharides isdifficult, time-consuming and expensive. A protein antigen which couldbe cheaply and easily produced would be an improvement.

[0011] If one were to make a polysaccharide-protein conjugate vaccine, aGBS-specific carrier protein may be preferable to one of the commonlyused carriers such as tetanus or diphtheria toxoids because of thepotential problems associated with some of these carrier proteins,particularly variable immunogenicity and the problems associated withrepeated vaccination with the same carrier protein. Selection ofappropriate carrier proteins is important for the development ofpolysaccharide-protein vaccine formulations. For example, Haemophilusinfluenzae type b poly- or oligosaccharide conjugated to differentprotein carriers has variable immunogenicity and elicits antibody withvarying avidity (Decker, M. D. et al (1992) J Pediatrics 120:184-189;Schlesinger, Y. (1992) JAMA 267:1489-1494). Repeated immunization withthe same carrier protein may also suppress immune responses bycompetition for specific B cells (epitopic suppression) or othermechanisms. This is of particular concern for the development of GBSvaccines since recently developed poly/oligosaccharide-protein conjugatevaccines against the bacteria H. influenzae, S. pneumoniae, and N.menizgitidis all utilize a restricted number of carrier proteins(tetanus toxoid, CRM197, diptheria toxoid), increasing the number ofexposures to these carriers an individual is likely to receive.Additionally, using tetanus as a carrier protein offers no specificadvantage beyond the improved immunogenicity of the vaccine. Asecond-generation vaccine containing a GBS-specific carrier proteinwould enhance immunogenicity and have an advantage in that aGBS-specific immune response would be generated against both the carrierprotein and the poly/oligosaccharide.

[0012] Therefore, in view of the aforementioned deficiencies attendantwith prior art vaccines and methods, it should be apparent that therestill exists a need in the art for an effective and immunogenic GBSvaccine. The availability and use of a GBS polypeptide in a conjugatevaccine is desirable. A GBS polypeptide which is present or expressed inall GBS serotypes would have the added advantage of providing broad,general immunity across many GBS serotypes. It would be particularlyrelevant and useful to provide a streptococcal vaccine or immunogenwhich is expressed broadly in various streptococcal species, wherebybroad or general immunity against multiple and unique groups ofstreptococci (for instance, Group A, Group B and S. pneumoniae),particularly against distinct virulent and clinically relevantstreptococcal bacteria, could thereby be generated.

[0013] The citation of references herein shall not be construed as anadmission that such is prior art to the present invention.

SUMMARY OF THE INVENTION

[0014] In accordance with the present invention, streptococcalpolypeptides termed extracellular matrix adhesins (Ema) are providedwhich are particularly useful in the identification and prevention ofinfections by streptococci.

[0015] In its broadest aspect, the present invention encompassesisolated polypeptides comprising an amino acid sequence of astreptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE. The isolated peptides, including combinations of one ormore thereof, are suitable for use in immunizing animals and humansagainst bacterial infection, particularly streptococci.

[0016] The present invention is directed to an isolated streptococcalEmaA polypeptide which comprises the amino acid sequence set out in SEQID NO: 2, and analogs, variants and immunogenic fragments thereof.

[0017] The present invention is directed to an isolated streptococcalEmaB polypeptide which comprises the amino acid sequence set out in SEQID NO: 4, and analogs, variants and immunogenic fragments thereof.

[0018] The present invention is directed to an isolated streptococcalEmaC polypeptide which comprises the amino acid sequence set out in SEQID NO: 6, and analogs, variants and immunogenic fragments thereof.

[0019] The present invention is directed to an isolated streptococcalEmaD polypeptide which comprises the amino acid sequence set out in SEQID NO: 8, and analogs, variants and immunogenic fragments thereof.

[0020] The present invention is directed to an isolated streptococcalEmaE polypeptide which comprises the amino acid sequence set out in SEQID NO: 10, and analogs, variants and immunogenic fragments thereof.

[0021] The present invention also provides Ema polypeptide homologs fromdistinct bacterial species, particularly including distinctstreptococcal species, more particularly including Group Bstreptococcus, Group A streptococcus (particularly S. pyogenes) and S.pneumoniae. The present invention also provides Ema polypeptides fromadditional distinct bacterial species, particularly includingEnterococcus faecalis and Corynebacterium diptheriae. Nucleic acidsencoding Ema polypeptide homologs from distinct bacterial species arealso provided.

[0022] The present invention thus provides an isolated streptococcal Emapolypeptide comprising the amino acid sequence set out in SEQ ID NO:23.An isolated nucleic acid which encodes the streptococcal polypeptide setout in SEQ ID NO:23 is further provided.

[0023] The invention thus further provides an isolated streptococcal Emapolypeptide comprising the amino acid sequence set out in SEQ ID NO:26.An isolated nucleic acid which encodes the streptococcal polypeptide setout in SEQ ID NO:26 is further provided.

[0024] The present invention further provides an isolated streptococcalEma polypeptide comprising the amino acid sequence set out in SEQ IDNO:37. An isolated nucleic acid which encodes the streptococcalpolypeptide set out in SEQ ID NO:37 is further provided.

[0025] An enterococcal Ema polypeptide is further provided comprisingthe amino acid sequence set out in SEQ ID NO:29. An isolated isolatednucleic acid which encodes the enterococcal polypeptide set out in SEQID NO:29 is also provided.

[0026] The invention provides an isolated Corynebacterium Emapolypeptide comprising the amino acid sequence set out in SEQ ID NO: 32.Also provided is an isolated nucleic acid which encodes theCorynebacterium polypeptide set out in SEQ ID NO: 32.

[0027] The invention provides an isolated bacterial polypeptidecomprising the amino acid sequence TLLTCTPYMINS/THRLLVR/KG (SEQ ID NO:34), wherein the polypeptide is not isolated from Actinomyces.

[0028] The invention further provides an isolated streptococcalpolypeptide comprising the amino acid sequence TLLTCTPYMINS/THRLLVR/KG(SEQ ID NO: 34).

[0029] Also provided is an isolated bacterial polypeptide comprising theamino acid sequence TLVTCTPYGINTHRLLVTA (SEQ ID NO: 35).

[0030] The present invention includes an isolated bacterial polypeptidecomprising the amino acid sequence TLVTCTPYGVNTKRLLVRG (SEQ ID NO: 36).An isolated streptococcal polypeptide comprising the amino acid sequenceTLVTCTPYGVNTKRLLVRG (SEQ ID NO: 36) is also provided.

[0031] The invention further includes an isolated polypeptide having theamino acid sequence selected from the group of TLLTCTPYMINS/THRLLVR/KG(SEQ ID NO: 34), TLVTCTPYGINTHRLLVTA (SEQ ID) NO: 35), andTLVTCTPYGVNTKRLLVRG (SEQ ID NO: 36).

[0032] The present invention contemplates the use of the polypeptides ofthe present invention in diagnostic tests and methods for determiningand/or monitoring of streptococcal infection. Thus, the presentinvention provides an isolated Ema polypeptide, particularly selectedfrom the group of EmaA, EmaB, EmaC, EmaD and EmaE, labeled with adetectable label.

[0033] In the instance where a radioactive label, such as the isotopes³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and¹⁸⁶Re are used, known currently available counting procedures may beutilized. In the instance where the label is an enzyme, detection may beaccomplished by any of the presently utilized calorimetric,spectrophotometric, fluorospectrophotometric, amperometric or gasometrictechniques known in the art.

[0034] The present invention extends to an immunogenic Ema polypeptide,particularly selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE,or a fragment thereof. The present invention also extends to immunogenicEma polypeptides wherein such polypeptides comprise a combination of atleast one immunogenic Ema polypeptide, selected from the group of EmaA,EmaB, EmaC, EmaD and EmaE, or immunogenic polypeptide fragment thereof,and a GBS polypeptide selected from the group of Spb1, Spb2, C proteinalpha antigen, Rib, Lmb, C5a-ase, or immunogenic fragments thereof.

[0035] In a further aspect, the present invention extends to vaccinesbased on the Ema proteins described herein. The present inventionprovides a vaccine comprising one or more streptococcal polypeptidesselected from the group of EmaA, EmaB, EmaC, EmaD and EmaE, and apharmaceutically acceptable adjuvant. The present invention provides avaccine comprising one or more streptococcal polypeptides selected fromthe group of the polypeptide of SEQ ID NO: 23, 26, and 37, and apharmaceutically acceptable adjuvant.

[0036] The present invention further provides a streptococcal vaccinecomprising one or more Group B streptococcal polypeptides selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE, further comprising one ormore additional streptococcal antigens. The present invention furtherprovides a GBS vaccine comprising one or more Group B streptococcalpolypeptides selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE,further comprising one or more additional GBS antigens. In a particularembodiment, the GBS antigen is selected from the group of thepolypeptide Spbl or an immunogenic fragment thereof, the polypeptideSpb2 or an immunogenic fragment thereof, C protein alpha antigen or animmunogenic fragment thereof, Rib or an immunogenic fragment thereof.Lmb or an immunogenic fragment thereof, C5a-ase or an immunogenicfragment thereof and Group B streptococcal polysaccharides oroligosaccharides.

[0037] In another aspect, the invention is directed to a vaccine forprotection of an animal subject from infection with streptococcicomprising an immunogenic amount of one or more Ema polypeptide EmaA,EmaB, EmaC, EmaD or EmaE, or a derivative or fragment thereof. Such avaccine may contain the protein conjugated covalently to a GBS bacterialpolysaccharide or oligosaccharide or polysaccharide or oligosaccharidefrom one or more GBS serotypes.

[0038] In a still further aspect, the present invention provides animmunogenic composition comprising one of more streptococcalpolypeptides selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE,and a pharmaceutically acceptable adjuvant.

[0039] The present invention further provides an immunogenic compositioncomprising one or more Group B streptococcal polypeptide selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE, further comprising one ormore antigens selected from the group of the polypeptide Spb1 or animmunogenic fragment thereof, the polypeptide Spb2 or an immunogenicfragment thereof, C protein alpha antigen or an immunogenic fragmentthereof, Rib or an immunogenic fragment thereof. Lmb or an immunogenicfragment thereof, C5a-ase or an immunogenic fragment thereof, and GroupB streptococcal polysaccharides or oligosaccharides.

[0040] The invention further provides pharmaceutical compositions,vaccines, and diagnostic and therapeutic methods of use thereof.

[0041] The invention provides pharmaceutical compositions comprising abacterial Ema polypeptide and a pharmaceutically acceptable carrier. Theinvention provides pharmaceutical compositions comprising astreptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE, the polypeptide of SEQ ID NO:23, the polypeptide of SEQID NO: 26, the polypeptide of SEQ ID NO:37, and a pharmaceuticallyacceptable carrier. The invention provides pharmaceutical compositionscomprising a streptococcal polypeptide selected from the group of EmaA,EmaB, EmaC, EmaD and EmaE, and a pharmaceutically acceptable carrier.The present invention further provides pharmaceutical compositionscomprising one or more GBS Ema polypeptide, or a fragment thereof, incombination with one or more of GBS polypeptide Spb1, Spb2, C proteinalpha antigen, Rib, Lmb, C5a-ase, a Group B streptococcal polysaccharideor oligosaccharide vaccine, and an anti-streptococcal vaccine.

[0042] In a still further aspect, the present invention provides apurified antibody to a streptococcal polypeptide selected from the groupof EmaA, EmaB, EmaC, EmaD and EmaE. In a still further aspect, thepresent invention provides a purified antibody to a streptococcalpolypeptide selected from the group of the polypeptide of SEQ ID NO:23,the polypeptide of SEQ ID NO: 26, and the polypeptide of SEQ ID NO:37.

[0043] Antibodies against the isolated polypeptides of the presentinvention include naturally raised and recombinantly preparedantibodies. These may include both polyclonal and monoclonal antibodiesprepared by known genetic techniques, as well as bi-specific (chimeric)antibodies, and antibodies including other functionalities suiting themfor diagnostic use. Such antibodies can be used in immunoassays todiagnose infection with a particular strain or species of bacteria. Theantibodies can also be used for passive immunization to treat aninfection with streptococcal bacteria including Group B streptococcus,Group A streptococcus, and S. pneumoniae. These antibodies may also besuitable for modulating bacterial adherence and/or invasion includingbut not limited to acting as competitive agents.

[0044] The present invention provides a monoclonal antibody to astreptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE. The invention thereby extends to an immortal cell linethat produces a monoclonal antibody to a streptococcal poypeptideselected from the group of EmaA, EmaB, EmaC, EmaD and EmaE.

[0045] An antibody to a streptococcal Ema polypeptide EmaA, EmaB, EmaC,EmaD or EmaE labeled with a detectable label is further provided. Inparticular embodiments, the label may selected from the group consistingof an enzyme, a chemical which fluoresces, and a radioactive element.

[0046] The present invention provides a pharmaceutical compositioncomprising one or more antibodies to a streptococcal protein selectedfrom the group of EmaA, EmaB, EmaC, EmaD and EmaE, and apharmaceutically acceptable carrier. The invention further provides apharmaceutical composition comprising a combination of at least twoantibodies to Group B streptococcal proteins and a pharmaceuticallyacceptable carrier, wherein at least one antibody to a protein selectedfrom the group of EmaA, EmaB, EmaC, EmaD, and EmaE is combined with atleast one antibody to a protein selected from the group of Spb1, Spb2,Rib, Lmb, C5a-ase and a C protein alpha antigen.

[0047] The present invention also relates to isolated nucleic acids,such as recombinant DNA molecules or cloned genes, or degeneratevariants thereof, mutants, analogs, or fragments thereof, which encodethe isolated polypeptide of the present invention or which competitivelyinhibit the activity of the polypeptide. The present invention furtherrelates to isolated nucleic acids, such as recombinant DNA molecules orcloned genes, or degenerate variants thereof, mutants, analogs, orfragments thereof, which encode a bacterial Ema polypeptide. The presentinvention further relates to isolated nucleic acids, such as recombinantDNA molecules or cloned genes, or degenerate variants thereof, mutants,analogs, or fragments thereof, which encode a streptococcal Emapolypeptide. The present invention further relates to isolated nucleicacids, such as recombinant DNA molecules or cloned genes, or degeneratevariants thereof, mutants, analogs, or fragments thereof, which encode astreptococcal Ema polypeptide, particularly selected from the group ofEmaA, EmaB, EmaC, EmaD and EmaE. Preferably, the isolated nucleic acid,which includes degenerates, variants, mutants, analogs, or fragmentsthereof, has a sequence as set forth in SEQ ID NOS: 1, 3, 5, 7 or 9. Ina further embodiment of the invention, the DNA sequence of therecombinant DNA molecule or cloned gene may be operatively linked to anexpression control sequence which may be introduced into an appropriatehost. The invention accordingly extends to unicellular hosts transformedwith the cloned gene or recombinant DNA molecule comprising a DNAsequence encoding an Ema protein, particularly selected from the groupof EmaA, EmaB, EmaC, EmaD and EmaE, and more particularly, the DNAsequences or fragments thereof determined from the sequences set forthabove.

[0048] In a particular embodiment, the nucleic acid encoding the EmaApolypeptide has the sequence selected from the group comprising SEQ IDNO: 1; a sequence that hybridizes to SEQ ID NO: 1 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO: 1 or a sequence thathybridizes to SEQ ID NO: 1 under moderate stringency hybridizationconditions; degenerate variants thereof, alleles thereof; andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaA polypeptide has the sequence selected from thegroup comprising SEQ ID NO: 1; a sequence complementary to SEQ ID NO: 1;or a homologous sequence which is substantially similar to SEQ ID NO: 1.In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO: 1.

[0049] In a particular embodiment, the nucleic acid encoding the EmaBpolypeptide has the sequence selected from the group comprising SEQ IDNO:3; a sequence that hybridizes to SEQ ID NO:3 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:3 or a sequence thathybridizes to SEQ ID NO:3 under moderate stringency hybridizationconditions; degenerate variants thereof, alleles thereof, andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaB polypeptide has the sequence selected from thegroup comprising SEQ ID NO:3; a sequence complementary to SEQ ID NO:3;or a homologous sequence which is substantially similar to SEQ ID NO:3.In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:3.

[0050] In a particular embodiment, the nucleic acid encoding the EmaCpolypeptide has the sequence selected from the group comprising SEQ IDNO:5; a sequence that hybridizes to SEQ ID NO:5 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:5 or a sequence thathybridizes to SEQ ID NO:5 under moderate stringency hybridizationconditions; degenerate variants thereof, alleles thereof, andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaC polypeptide has the sequence selected from thegroup comprising SEQ ID NO:5; a sequence complementary to SEQ ID NO:5;or a homologous sequence which is substantially similar to SEQ ID NO:5.In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:5.

[0051] In a particular embodiment, the nucleic acid encoding the EmaDpolypeptide has the sequence selected from the group comprising SEQ IDNO:7; a sequence that hybridizes to SEQ ID NO:7 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:7 or a sequence thathybridizes to SEQ ID NO:7 under moderate stringency hybridizationconditions; degenerate variants thereof, alleles thereof, andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaD polypeptide has the sequence selected from thegroup comprising SEQ ID NO:7; a sequence complementary to SEQ ID NO:7;or a homologous sequence which is substantially similar to SEQ ID NO:7.In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:7.

[0052] In a particular embodiment, the nucleic acid encoding the EmaEpolypeptide has the sequence selected from the group comprising SEQ IDNO:9; a sequence that hybridizes to SEQ ID NO:9 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:9 or a sequence thathybridizes to SEQ ID NO:9 under moderate stringency hybridizationconditions; degenerate variants thereof; alleles thereof; andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaE polypeptide has the sequence selected from thegroup comprising SEQ ID NO:9; a sequence complementary to SEQ ID NO:9;or a homologous sequence which is substantially similar to SEQ ID NO:9In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:9.

[0053] In a further embodiment, the nucleic acid encoding the bacterialEma polypeptide comprises the sequence selected from the groupcomprising SEQ ID NO: 24, 27, 30 and 33. In a further embodiment, thenucleic acid encoding the bacterial Ema polypeptide has the sequenceselected from the group comprising SEQ ID NO: 24, 27, 30 and 33.

[0054] A nucleic acid capable of encoding a streptococcal polypeptideEmaA, EmaB, EmaC, EmaD or EmaE which is a recombinant DNA molecule isfurther provided. Such a recombinant DNA molecule wherein the DNAmolecule is operatively linked to an expression control sequence is alsoprovided herein.

[0055] The present invention relates to nucleic acid vaccines or DNAvaccines comprising nucleic acids encoding immunogenic streptococcal Emapolypeptides, particularly selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE. The present invention relates to nucleic acid vaccines orDNA vaccines comprising nucleic acids encoding one or more immunogenicEma polypeptide or a fragment thereof or any combination of one or moreEma polypeptide EmaA, EmaB, EmaC, EmaD or EmaE with at least one otherpolypeptide, particularly a GBS polypeptide, more particularly whereinsaid other GBS polypeptide is selected from the group of Spb1, Spb2, Cprotein alpha antigen, Rib, Lmb, C5a-ase, and immunogenic polypeptidefragments thereof.

[0056] The invention further relates to a vaccine for protection of ananimal subject from infection with a streptococcal bacterium comprisinga vector containing a gene encoding an Ema polypeptide selected from thegroup of EmaA, EmaB, EmaC, EmaD and EmaE operatively associated with apromoter capable of directing expression of the gene in the subject. Thepresent invention further provides a nucleic acid vaccine comprising arecombinant DNA molecule capable of encoding a GBS polypeptide EmaA,EmaB, EmaC, EmaD or EmaE.

[0057] The invention further relates to a vaccine for protection of ananimal subject from infection with a Group B streptococcal bacteriumcomprising a vector containing a gene encoding an Ema polypeptideselected from the group of EmaA, EmaB, EmaC, EmaD and EmaE operativelyassociated with a promoter capable of directing expression of the genein the subject. The present invention further provides a nucleic acidvaccine comprising a recombinant DNA molecule capable of encoding a GBSpolypeptide EmaA, EmaB, EmaC, EmaD or EmaE.

[0058] The present invention provides a vector which comprises thenucleic acid capable of encoding encoding an Ema polypeptide selectedfrom the group of EmaA, EmaB, EmaC, EmaD and EmaE and a promoter. Thepresent invention provides a vector which comprises the nucleic acid ofany of SEQ ID NO: 1, 3, 5, 7 or 9 and a promoter. The inventioncontemplates a vector wherein the promoter comprises a bacterial, yeast,insect or mammalian promoter. The invention contemplates a vectorwherein the vector is a plasmid, cosmid, yeast artificial chromosome(YAC), bacteriophage or eukaryotic viral DNA.

[0059] The present invention further provides a host vector system forthe production of a polypeptide which comprises the vector capable ofencoding an Ema polypeptide, particularly selected from the group ofEmaA, EmaB, EmaC, EmaD and EmaE in a suitable host cell. A host vectorsystem is provided wherein the suitable host cell comprises aprokaryotic or eukaryotic cell. A unicellular host transformed with arecombinant DNA molecule or vector capable of encoding encoding an Emapolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaEis thereby provided.

[0060] The present invention includes methods for determining andmonitoring infection by streptococci by detecting the presence of astreptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE. In a particular such method, the streptococcal Emapolypeptide is measured by:

[0061] a. contacting a sample in which the presence or activity of aStreptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE is suspected with an antibody to the said streptococcalpolypeptide under conditions that allow binding of the streptococcalpolypeptide to the antibody to occur; and

[0062] b. detecting whether binding has occurred between thestreptococcal polypeptide from the sample and the antibody;

[0063] wherein the detection of binding indicates the presence oractivity of the streptococcal polypeptide in the sample.

[0064] The present invention includes methods for determining andmonitoring infection by streptococci by detecting the presence of astreptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE. In a particular such method, the streptococcal Emapolypeptide is measured by:

[0065] a. contacting a sample in which the presence or activity of aStreptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE is suspected with an antibody to the said streptococcalpolypeptide under conditions that allow binding of the streptococcalpolypeptide to the antibody to occur; and

[0066] b. detecting whether binding has occurred between thestreptococcal polypeptide from the sample and the antibody;

[0067] wherein the detection of binding indicates the presence oractivity of the streptococcal polypeptide in the sample.

[0068] The present invention includes methods for determining andmonitoring infection by Group B streptococci by detecting the presenceof a Group B streptococcal polypeptide selected from the group of EmaA,EmaB, EmaC, EmaD and EmaE. In a particular such method, thestreptococcal Ema polypeptide is measured by:

[0069] a. contacting a sample in which the presence or activity of aGroup B streptococcal polypeptide selected from the group of EmaA, EmaB,EmaC, EmaD and EmaE is suspected with an antibody to the said Group Bstreptococcal polypeptide under conditions that allow binding of theGroup B streptococcal polypeptide to the antibody to occur; and

[0070] b. detecting whether binding has occurred between the Group Bstreptococcal polypeptide from the sample and the antibody;

[0071] wherein the detection of binding indicates the presence oractivity of the Group B streptococcal polypeptide in the sample.

[0072] The present invention further provides a method for detecting thepresence of a bacterium having a gene encoding a streptococcalpolypeptide selected from the group of emaA, emaB, emaC, emaD and emaE,comprising:

[0073] a. contacting a sample in which the presence or activity of thebacterium is suspected with an oligonucleotide which hybridizes to astreptococcal polypeptide gene selected from the group of emaA, emaB,emaC, emaD and emaE, under conditions that allow specific hybridizationof the oligonucleotide to the gene to occur; and

[0074] b. detecting whether hybridization has occurred between theoligonucleotide and the gene;

[0075] wherein the detection of hybridization indicates that presence oractivity of the bacterium in the sample.

[0076] The invention includes an assay system for screening of potentialcompounds effective to modulate the activity of a streptococcal proteinEmaA, EmaB, EmaC, EmaD or EmaE of the present invention. In oneinstance, the test compound, or an extract containing the compound,could be administered to a cellular sample expressing the particular Emaprotein to determine the compound's effect upon the activity of theprotein by comparison with a control. In a further instance the testcompound, or an extract containing the compound, could be administeredto a cellular sample expressing the Ema protein to determine thecompound's effect upon the activity of the protein, and thereby onadherence of said cellular sample to host cells, by comparison with acontrol.

[0077] It is still a further object of the present invention to providea method for the prevention or treatment of mammals to control theamount or activity of streptococci, so as to treat or prevent theadverse consequences of invasive, spontaneous, or idiopathicpathological states.

[0078] It is still a further object of the present invention to providea method for the prevention or treatment of mammals to control theamount or activity of Group B streptococci, so as to treat or preventthe adverse consequences of invasive, spontaneous, or idiopathicpathological states.

[0079] The invention provides a method for preventing infection with abacterium that expresses a streptococcal Ema polypeptide comprisingadministering an immunogenically effective dose of a vaccine comprisingan Ema polypeptide selected from the group of EmaA, EmaB, EmaC, EmaD andEmaE to a subject.

[0080] The invention further provides a method for preventing infectionwith a bacterium that expresses a Group B streptococcal Ema polypeptidecomprising administering an immunogenically effective dose of a vaccinecomprising an Ema polypeptide selected from the group of EmaA, EmaB,EmaC, EmaD and EmaE to a subject.

[0081] The present invention is directed to a method for treatinginfection with a bacterium that expresses a streptococcal Emapolypeptide comprising administering a therapeutically effective dose ofa pharmaceutical composition comprising an Ema polypeptide selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE, and a pharmaceuticallyacceptable carrier to a subject.

[0082] The invention further provides a method for treating infectionwith a bacterium that expresses a streptococcal Ema polypeptidecomprising administering a therapeutically effective dose of apharmaceutical composition comprising an antibody to an Ema polypeptideselected from the group of EmaA, EmaB, EmaC, EmaD and EmaE, and apharmaceutically acceptable carrier to a subject.

[0083] In a further aspect, the invention provides a method of inducingan immune response in a subject which has been exposed to or infectedwith a streptococcal bacterium comprising administering to the subjectan amount of the pharmaceutical composition comprising an Emapolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE,and a pharmaceutically acceptable carrier, thereby inducing an immuneresponse.

[0084] The invention still further provides a method for preventinginfection by a streptococcal bacterium in a subject comprisingadministering to the subject an amount of a pharmaceutical compositioncomprising an antibody to an Ema polypeptide selected from the group ofEmaA, EmaB, EmaC, EmaD and EmaE and a pharmaceutically acceptablecarrier or diluent, thereby preventing infection by a streptococcalbacterium.

[0085] In a further aspect, the invention provides a method of inducingan immune response in a subject which has been exposed to or infectedwith a Group B streptococcal bacterium comprising administering to thesubject an amount of the pharmaceutical composition comprising an Emapolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE,and a pharmaceutically acceptable carrier, thereby inducing an immuneresponse.

[0086] The invention still further provides a method for preventinginfection by a Group B streptococcal bacterium in a subject comprisingadministering to the subject an amount of a pharmaceutical compositioncomprising an antibody to an Ema polypeptide selected from the group ofEmaA, EmaB, EmaC, EmaD and EmaE and a pharmaceutically acceptablecarrier or diluent, thereby preventing infection by a streptococcalbacterium.

[0087] The invention further provides an ema mutant bacteria which isnon-adherent and/or non-invasive to cells, particularly which is mutatedin one or more genes selected from the group of emaA, emaB, emaC, emaDand emaE. Particularly, such ema mutant is a streptococcal bacteria.More particularly, such ema mutant is a Group B streptococcal bacteria.Such non-adherent and/or non-invasive ema mutant bacteria can further beutilized in expressing other immunogenic or therapeutic proteins for thepurposes of eliciting immune responses to any such other proteins in thecontext of vaccines and in other forms of therapy.

[0088] Other objects and advantages will become apparent to thoseskilled in the art from a review of the following description whichproceeds with reference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0089]FIG. 1 depicts the restriction digest pattern (RDP) type III-3specific probes. Dot blot hybridization of probe DY1-1 with genomic DNAisolated from type III GBS. 10 ug of genomic DNA from each of 62 typeIII GBS strains was transferred to nylon membrane. Radiolabeled probeDY1-1 hybridized with DNA from all III-3 strains (rows A-D) includingthe original type III-3 strain (well E-1). The probe failed to hybridizewith DNA from III-2 strains (F1-F10, G1-7) including the original strainused in the subtraction hybridization (well E 10) and III-1 strains(wells H1-3; cf FIG. 3). The same pattern of hybridization was observedusing probe DY1-11.

[0090]FIG. 2 depicts the nucleic acid and predicted amino acid sequenceof emaA.

[0091]FIG. 3 depicts the nucleic acid and predicted amino acid sequenceof emaB.

[0092]FIG. 4 depicts the nucleic acid and predicted amino acid sequenceof emaC.

[0093]FIG. 5 depicts the nucleic acid and predicted amino acid sequenceof emaD.

[0094] FIGS. 6A-D depicts the nucleic acid and predicted amino acidsequence of emaE.

DETAILED DESCRIPTION

[0095] The present invention provides novel Group B streptococcal Emapolypeptides and their Ema homologs in distinct bacterial species,including distinct streptococcal species. The present invention relatesto novel streptococcal Ema polypeptides, particularly selected from thegroup of EmaA, EmaB, EmaC, EmaD and EmaE, and fragments thereof. Nucleicacids encoding Ema polypeptides, and diagnostic and therapeuticcompositions and methods based thereon for identification and preventionof infections by virulent forms of streptococci are provided. Inparticular, the present invention includes Group B streptococcal Emapolypeptides. The invention further includes polypeptide homologs of theGBS Ema polypeptides, particularly streptococcal homologs, moreparticularly Ema homologs of S. pneumoniae and S. pyogenes. BacterialEma polypeptide homologs in E. faecalis and C. diptheriae are alsoprovided.

Polypeptides

[0096] The present invention is directed to an isolated polypeptidecomprising an amino acid sequence of a bacterial Ema polypeptide.Bacterial Ema polypepties are provided from streptococcus, enterococcusand corynebacterium. The present invention is particularly directed toan isolated polypeptide comprising an amino acid sequence of astreptococcal Ema polypeptide selected from the group of EmaA, EmaB,EmaC, EmaD and EmaE. The present invention is particularly directed toan isolated polypeptide comprising an amino acid sequence of a Groupstreptococcal Ema polypeptide selected from the group of EmaA, EmaB,EmaC, EmaD and EmaE. Additional S. pneumoniae and S. pyogenes Emapolypeptides are included in the invention. E. faecalis and C.diptheriae Ema polypeptides are also included in the invention.

[0097] The polypeptides of the present invention are suitable for use inimmunizing animals broadly against streptococcal infection. Thepolypeptides of the present invention are suitable for use in immunizinganimals broadly against Group B, Group A, and S. pneumoniaestreptococcal infection. The polypeptides of the present invention aresuitable for use in immunizing animals against Group B streptococci.These polypeptide or peptide fragments thereof, when formulated with anappropriate adjuvant, are used in vaccines for protection againststreptococci, particularly Group B streptococci, and against otherbacteria with cross-reactive proteins.

[0098] GBS proteins with streptococcal homologs outside of Group B havebeen previously identified (Lachenauer C S and Madoff L C (1997) Adv ExpMed Biol. 418:615-8; Brady L. J. et al (1991) Infect Immun59(12):4425-35; Stahlhammer-Carlemalm M. et al (2000) J Infect Dis182(1):142-129). Stahlhammer-Carlemalm et al have demonstratedcross-protection between Group A and Group B streptococci due tocross-reacting surface proteins (Stahlharnmer-Carlemalm M. et al (2000)J Infect Dis 182(1): 142-129). The R28 protein of group A streptococcus(GAS) and the Rib protein of group B streptococcus (GBS) are surfacemolecules that elicit protective immunity to experimental infection.These proteins are members of the same family and cross-reactimmunologically. In spite of extensive amino acid residue identity, thecross-reactivity between R28 and Rib was found to be limited, as shownby analysis with highly purified proteins and specific antisera.Nevertheless, immunization of mice with purified R28 conferredprotection against lethal infection with Rib-expressing GBS strains, andimmunization with Rib conferred protection against R28-expressing GAS.Thus, R28 and Rib elicited cross-protective immunity.

[0099] The present invention is directed to an isolated streptococcalEmaA polypeptide which comprises the amino acid sequence set out in SEQID NO: 2, and analogs, variants and immunogenic fragments thereof.

[0100] The present invention is directed to an isolated streptococcalEmaB polypeptide which comprises the amino acid sequence set out in SEQID NO: 4, and analogs, variants and immunogenic fragments thereof.

[0101] The present invention is directed to an isolated streptococcalEmaC polypeptide which comprises the amino acid sequence set out in SEQID NO: 6, and analogs, variants and immunogenic fragments thereof.

[0102] The present invention is directed to an isolated streptococcalEmaD polypeptide which comprises the amino acid sequence set out in SEQID NO: 8, and analogs, variants and immunogenic fragments thereof.

[0103] The identity or location of one or more amino acid residues maybe changed or modified to include variants such as, for example,deletions containing less than all of the residues specified for theprotein, substitutions wherein one or more residues specified arereplaced by other residues and additions wherein one or more amino acidresidues are added to a terminal or medial portion of the polypeptide.These molecules include: the incorporation of codons “preferred” forexpression by selected non-mammalian hosts; the provision of sites forcleavage by restriction endonuclease enzymes; and the provision ofadditional initial, terminal or intermediate DNA sequences thatfacilitate construction of readily expressed vectors.

[0104] The present invention is directed to an isolated Group Bstreptococcal EmaE polypeptide which comprises the amino acid sequenceset out in SEQ ID NO: 10, and analogs, variants and immunogenicfragments thereof.

[0105] The present invention thus provides an isolated streptococcal Emapolypeptide comprising the amino acid sequence,set out in SEQ ID NO:23.An isolated nucleic acid which encodes the streptococcal polypeptide setout in SEQ ID NO:23 is further provided.

[0106] The invention thus further provides an isolated streptococcal Emapolypeptide comprising the amino acid sequence set out in SEQ ID NO:26.An isolated nucleic acid which encodes the streptococcal polypeptide setout in SEQ ID NO:26 is further provided.

[0107] The present invention further provides an isolated streptococcalEma polypeptide comprising the amino acid sequence set out in SEQ IDNO:37. An isolated nucleic acid which encodes the streptococcalpolypeptide set out in SEQ ID NO:37 is further provided.

[0108] An enterococcal Ema polypeptide is further provided comprisingthe amino acid sequence set out in SEQ ID NO:29. An isolated isolatednucleic acid which encodes the enterococcal polypeptide set out in SEQID NO:29 is also provided.

[0109] The invention provides an isolated Corynebacterium Emapolypeptide comprising the amino acid sequence set out in SEQ ID NO: 32.Also provided is an isolated nucleic acid which encodes theCorynebacterium polypeptide set out in SEQ ID NO: 32.

[0110] The invention provides an isolated bacterial polypeptidecomprising the amino acid sequence TLLTCTPYMINS/THRLL VR/KG (SEQ ID NO:34), wherein the polypeptide is not isolated from Actinomyces.

[0111] The invention further provides an isolated streptococcalpolypeptide comprising the amino acid sequence TLLTCTPYMINS/THRLL VR/KG(SEQ ID NO: 34).

[0112] Also provided is an isolated bacterial polypeptide comprising theamino acid sequence TLVTCTPYGINTHRLLVTA (SEQ ID NO: 35).

[0113] The present invention includes an isolated bacterial polypeptidecomprising the amino acid sequence TLVTCTPYGVNTKRLLVRG (SEQ ID NO: 36).An isolated streptococcal polypeptide comprising the amino acid sequenceTLVTCTPYGVNTKRLLVRG (SEQ ID NO: 36) is also provided.

[0114] The invention further includes an isolated polypeptide having theamino acid sequence selected from the group of TLLTCTPYMINS/THRLLVR/KG(SEQ ID NO: 34), TLVTCTPYGINTHRLLVTA (SEQ ID NO: 35), andTLVTCTPYGVNTKRLLVRG (SEQ ID NO: 36).

[0115] The present invention contemplates the use of the streptococcalpolypeptides of the present invention in diagnostic tests and methodsfor determining and/or monitoring of streptococcal infection. Thus, thepresent invention provides an isolated GBS Ema polypeptide, particularlyselected from the group of EmaA, EmaB, EmaC, EmaD and EmaE, labeled witha detectable label.

[0116] In the instance where a radioactive label, such as the isotopes³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹ Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, 90Y, ¹²⁵I, ¹³¹I, and¹⁸⁶Re are used, known currently available counting procedures may beutilized. In the instance where the label is an enzyme, detection may beaccomplished by any of the presently utilized colorimetric,spectrophotometric, fluorospectrophotometric, amperometric or gasometrictechniques known in the art.

[0117] The present invention extends to an immunogenic bacterial Emapolypeptide. The present invention extends to an immunogenicstreptococcal Ema polypeptide, particularly selected from the group ofEmaA, EmaB, EmaC, EmaD and EmaE, or a fragment thereof. The presentinvention also extends to immunogenic GBS Ema polypeptides wherein suchpolypeptides comprise a combination of at least one immunogenic GBS Emapolypeptide, selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE,or immunogenic polypeptide fragment thereof and GBS polypeptide Spb1,Spb2, C protein alpha antigen, Rib or immunogenic fragments thereof.

[0118] As defined herein, “adhesion” means noncovalent binding of abacteria to a human cell or secretion that is stable enough to withstandwashing.

[0119] The term “extracellular matrix adhesin”, “Ema”, “ema” and anyvariants not specifically listed, may be used herein interchangeably,and as used throughout the present application and claims refer toproteinaceous material including single or multiple proteins, andextends to those proteins having the amino acid sequence data describedherein and particularly identified by (SEQ ID NOS: 2, 4, 6, 8, 10, 23,26, 29, 32 and 37), and the profile of activities set forth herein andin the claims. In particular the Ema proteins provided herein includeEmaA, EmaB, EmaC, EmaD and EmaE. The Ema proteins include bacterial Emahomologs. Bacterial Ema homologs include those from streptococcalspecies and other bacterial species. Accordingly, proteins andpolypeptides displaying substantially equivalent or altered activity arelikewise contemplated. These modifications may be deliberate, forexample, such as modifications obtained through site-directedmutagenesis, or may be accidental, such as those obtained throughmutations in hosts that are producers of one or more Ema polypeptide.Also, the term “extracellular matrix adhesin (Ema)” is intended toinclude within its scope proteins specifically recited herein as well asall substantially homologous analogs and allelic variations.

[0120] This invention provides an isolated immunogenic polypeptidecomprising an amino acid sequence of a bacterial Ema polypeptide. Thisinvention provides an isolated immunogenic polypeptide comprising anamino acid sequence of a streptococcal Ema polypeptide, particularlyselected from the group of EmaA, EmaB, EmaC, EmaD and EmaE. It iscontemplated by this invention that the immunogenic polypeptide has theamino acid sequence set forth in any of SEQ ID NOS: 2, 4, 6, 8, 10, 23,26, 29, 32 and 37, including immunogenic fragments, mutants, variants,analogs, or derivatives, thereof.

[0121] This invention is directed to analogs of the polypeptide whichcomprise the amino acid sequence as set forth above. The analogpolypeptide may have an N-terminal methionine or a polyhistidineoptionally attached to the N or COOH terminus of the polypeptide whichcomprise the amino acid sequence.

[0122] In another embodiment, this invention contemplates peptidefragments of the polypeptide which result from proteolytic digestionproducts of the polypeptide. In another embodiment, the derivative ofthe polypeptide has one or more chemical moieties attached thereto. Inanother embodiment the chemical moiety is a water soluble polymer. Inanother embodiment the chemical moiety is polyethylene glycol. Inanother embodiment the chemical moiety is mon-, di-, tri- ortetrapegylated. In another embodiment the chemical moiety is N-terminalmonopegylated.

[0123] Attachment of polyethylene glycol (PEG) to compounds isparticularly useful because PEG has very low toxicity in mammals(Carpenter et al., 1971). For example, a PEG adduct of adenosinedeaminase was approved in the United States for use in humans for thetreatment of severe combined immunodeficiency syndrome. A secondadvantage afforded by the conjugation of PEG is that of effectivelyreducing the immunogenicty and antigenicity of heterologous compounds.For example, a PEG adduct of a human protein might be useful for thetreatment of disease in other mammalian species without the risk oftriggering a severe immune response. The compound of the presentinvention may be delivered in a microencapsulation device so as toreduce or prevent an host immune response against the compound oragainst cells which may produce the compound. The compound of thepresent invention may also be delivered microencapsulated in a membrane,such as a liposome.

[0124] Numerous activated forms of PEG suitable for direct reaction withproteins have been described. Useful PEG reagents for reaction withprotein amino groups include active esters of carboxylic acid orcarbonate derivatives, particularly those in which the leaving groupsare N-hydroxysuccinimide, p-nitrophenol, imidazole or1-hydroxy-2nitrobenzene4-sulfonate. PEG derivatives containing maleimidoor haloacetyl groups are useful reagents for the modification of proteinfree sulfhydryl groups. Likewise, PEG reagents containing aminohydrazine or hydrazide groups are useful for reaction with aldehydesgenerated by periodate oxidation of carbohydrate groups in proteins.

[0125] In one embodiment, the amino acid residues of the polypeptidedescribed herein are preferred to be in the “L” isomeric form. Inanother embodiment, the residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional property of lectin activity is retained by the polypeptide.NH₂ refers to the free amino group present at the amino terminus of apolypeptide. COOH refers to the free carboxy group present at thecarboxy terminus of a polypeptide. Abbreviations used herein are inkeeping with standard polypeptide nomenclature, J. Biol. Chem.,243:3552-59 (1969).

[0126] It should be noted that all amino-acid residue sequences arerepresented herein by formulae whose left and right orientation is inthe conventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues.

[0127] Synthetic polypeptide, prepared using the well known techniquesof solid phase, liquid phase, or peptide condensation techniques, or anycombination thereof, can include natural and unnatural amino acids.Amino acids used for peptide synthesis may be standard Boc (N^(α)-aminoprotected N^(α)-t-butyloxycarbonyl) amino acid resin with the standarddeprotecting, neutralization, coupling and wash protocols of theoriginal solid phase procedure of Merrifield (1963, J. Am. Chem. Soc.85:2149-2154), or the base-labile N^(α)-amino protected9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpinoand Han (1972, J. Org. Chem. 37:3403-3409). Thus, polypeptide of theinvention may comprise D-amino acids, a combination of D- and L-aminoacids, and various “designer” amino acids (e.g, β-methyl amino acids,Cα-methyl amino acids, and Nα-methyl amino acids, etc.) to conveyspecial properties. Synthetic amino acids include omithine for lysine,fluorophenylalanine for phenylalanine, and norleucine for leucine orisoleucine. Additionally, by assigning specific amino acids at specificcoupling steps, α-helices, β turns, β sheets, γ-turns, and cyclicpeptides can be generated.

[0128] In one aspect of the invention, the peptides may comprise aspecial amino acid at the C-terminus which incorporates either a CO₂H orCONH₂ side chain to simulate a free glycine or a glycine-amide group.Another way to consider this special residue would be as a D or L aminoacid analog with a side chain consisting of the linker or bond to thebead. In one embodiment, the pseudo-free C-terminal residue may be ofthe D or the L optical configuration; in another embodiment, a racemicmixture of D and L-isomers may be used.

[0129] In an additional embodiment, pyroglutamate may be included as theN-terminal residue of the peptide. Although pyroglutamate is notamenable to sequence by Edman degradation, by limiting substitution toonly 50% of the peptides on a given bead with N-terminal pyroglutamate,there will remain enough non-pyroglutamate peptide on the bead forsequencing. One of ordinary skill would readily recognize that thistechnique could be used for sequencing of any peptide that incorporatesa residue resistant to Edman degradation at the N-terminus. Othermethods to characterize individual peptides that demonstrate desiredactivity are described in detail infra. Specific activity of a peptidethat comprises a blocked N-terminal group, e.g., pyroglutamate, when theparticular N-terminal group is present in 50% of the peptides, wouldreadily be demonstrated by comparing activity of a completely (100%)blocked peptide with a non-blocked (0%) peptide.

[0130] In addition, the present invention envisions preparing peptidesthat have more well defined structural properties, and the use ofpeptidomimetics, and peptidomimetic bonds, such as ester bonds, toprepare peptides with novel properties. In another embodiment, a peptidemay be generated that incorporates a reduced peptide bond, i.e.,R₁—CH₂—NH—R₂, where R₁ and R₂ are amino acid residues or sequences. Areduced peptide bond may be introduced as a dipeptide subunit. Such amolecule would be resistant to peptide bond hydrolysis, e.g., proteaseactivity. Such peptides would provide ligands with unique function andactivity, such as extended half-lives in vivo due to resistance tometabolic breakdown, or protease activity. Furthermore, it is well knownthat in certain systems constrained peptides show enhanced functionalactivity (Hruby, 1982, Life Sciences 31:189-199; Hruby et al., 1990,Biochem J. 268:249-262); the present invention provides a method toproduce a constrained peptide that incorporates random sequences at allother positions.

[0131] A constrained, cyclic or rigidized peptide may be preparedsynthetically, provided that in at least two positions in the sequenceof the peptide an amino acid or amino acid analog is inserted thatprovides a chemical functional group capable of cross-linking toconstrain, cyclise or rigidize the peptide after treatment to form thecross-link. Cyclization will be favored when a turn-inducing amino acidis incorporated. Examples of amino acids capable of cross-linking apeptide are cysteine to form disulfide, aspartic acid to form a lactoneor a lactase, and a chelator such as γ-carboxyl-glutamic acid (Gla)(Bachem) to chelate a transition metal and form a crosslink. Protectedγ-carboxyl glutamic acid may be prepared by modifying the synthesisdescribed by Zee-Cheng and Olson (1980, Biophys. Biochem. Res. Commun.94:1128-1132). A peptide in which the peptide sequence comprises atleast two amino acids capable of cross-linking may be treated, e.g., byoxidation of cysteine residues to form a disulfide or addition of ametal ion to form a chelate, so as to crosslink the peptide and form aconstrained, cyclic or rigidized peptide.

[0132] The present invention provides strategies to systematicallyprepare cross-links. For example, if four cysteine residues areincorporated in the peptide sequence, different protecting groups may beused (Hiskey, 1981, in The Peptides: Analysis, Synthesis, Biology, Vol.3, Gross and Meienhofer, eds., Academic Press: New York, pp. 137167;Ponsanti et al., 1990, Tetrahedron 46:8255-8266). The first pair ofcysteine may be deprotected and oxidized, then the second set may bedeprotected and oxidized. In this way a defined set of disulfidecross-links may be formed. Alternatively, a pair of cysteine and a pairof collating amino acid analogs may be incorporated so that thecross-links are of a different chemical nature.

[0133] The following non-classical amino acids may be incorporated inthe peptide in order to introduce particular conformational motifs:1,2,3,4-tetrahydroisoquinoline-3carboxylate (Kazmierski et al., 1991, J.Am. Chem. Soc. 113:2275-2283); (2S,3S)-methyl-phenylalanine,(2S,3R)-methyl-phenylalanine, (2R,3 S)-methyl-phenylalanine and(2R,3R)-methyl-phenylalanine (Kazmierski and Hruby, 1991, TetrahedronLett.); 2-aminotetrahydronaphthalene-2-carboxylic acid (Landis, 1989,Ph.D. Thesis, University of Arizona);hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al.,1989, J. Takeda Res. Labs. 43:53-76); β-carboline (D and L) (Kazmierski,1988, Ph.D. Thesis, University of Arizona); HIC (histidine isoquinolinecarboxylic acid) (Zechel et al., 1991, Int. J. Pep. Protein Res. 43);and HIC (histidine cyclic urea) (Dharanipragada).

[0134] The following amino acid analogs and peptidomimetics may beincorporated into a peptide to induce or favor specific secondarystructures: LL-Acp (LL-3-amino2-propenidone-6-carboxylic acid), a β-turninducing dipeptide analog (Kemp et al., 1985, J. Org. Chem.50:5834-5838); β-sheet inducing analogs (Kemp et al., 1988, TetrahedronLett. 29:5081-5082); β-turn inducing analogs (Kemp et al., 1988,Tetrahedron Lett. 29:5057-5060); ∝-helix inducing analogs (Kemp et al.,1988, Tetrahedron Lett. 29:4935-4938); γ-turn inducing analogs (Kemp etal., 1989, J. Org. Chem. 54:109:115); and analogs provided by thefollowing references: Nagai and Sato, 1985, Tetiahedron Lett.26:647-650; DiMaio et al., 1989, J. Chem. Soc. Perkin Trans. p. 1687;also a Gly-Ala turn analog (Kahn et al., 1989, Tetrahedron Lett.30:2317); amide bond isostere (Jones et al., 1988, Tetrahedron Lett.29:3853-3856); tretrazol (Zabrocki et al., 1988, J. Am. Chem. Soc.110:5875-5880); DTC (Samanen et al., 1990, Int. J. Protein Pep. Res.35:501:509); and analogs taught in Olson et al., 1990, J. Am. Chem. Sci.112:323-333 and Garvey et al., 1990, J. Org. Chem. 56:436.Conformationally restricted mimetics of beta turns and beta bulges, andpeptides containing them, are described in U.S. Pat. No. 5,440,013,issued Aug. 8, 1995 to Kahn.

[0135] The present invention further provides for modification orderivatization of the polypeptide or peptide of the invention.Modifications of peptides are well known to one of ordinary skill, andinclude phosphorylation, carboxymethylation, and acylation.Modifications may be effected by chemical or enzymatic means. In anotheraspect, glycosylated or fatty acylated peptide derivatives may beprepared. Preparation of glycosylated or fatty acylated peptides is wellknown in the art. Fatty acyl peptide derivatives may also be prepared.For example, and not by way of limitation, a free amino group(N-terminal or lysyl) may be acylated, e.g., myristoylated. In anotherembodiment an amino acid comprising an aliphatic side chain of thestructure —(CH₂)_(n)CH₃ may be incorporated in the peptide. This andother peptide-fatty acid conjugates suitable for use in the presentinvention are disclosed in U.K. Patent GB-8809162.4, InternationalPatent Application PCT/AU89/00166, and reference 5, supra.

[0136] Chemical Moieties For Derivatization. Chemical moieties suitablefor derivatization may be selected from among water soluble polymers.The polymer selected should be water soluble so that the component towhich it is attached does not precipitate in an aqueous environment,such as a physiological environment. Preferably, for therapeutic use ofthe end-product preparation, the polymer will be pharmaceuticallyacceptable. One skilled in the art will be able to select the desiredpolymer based on such considerations as whether the polymer/componentconjugate will be used therapeutically, and if so, the desired dosage,circulation time, resistance to proteolysis, and other considerations.For the present component or components, these may be ascertained usingthe assays provided herein.

[0137] The water soluble polymer may be selected from the groupconsisting of, for example, polyethylene glycol, copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols and polyvinyl alcohol. Polyethylene glycol propionaldenhyde mayhave advantages in manufacturing due to its stability in water.

[0138] The polymer may be of any molecular weight, and may be branchedor unbranched. For polyethylene glycol, the preferred molecular weightis between about 2 kDa and about 100 kDa (the term “about” indicatingthat in preparations of polyethylene glycol, some molecules will weighmore, some less, than the stated molecular weight) for ease in handlingand manufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

[0139] The number of polymer molecules so attached may vary, and oneskilled in the art will be able to ascertain the effect on function Onemay mono-derivative, or may provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of polyethyleneglycols). The proportion of polymer molecules to component or componentsmolecules will vary, as will their concentrations in the reactionmixture. In general, the optimum ratio (in terms of efficiency ofreaction in that there is no excess unreacted component or componentsand polymer) will be determined by factors such as the desired degree ofderivatization (e.g., mono, di-, tri-, etc.), the molecular weight ofthe polymer selected, whether the polymer is branched or unbranched, andthe reaction conditions.

[0140] The polyethylene glycol molecules (or other chemical moieties)should be attached to the component or components with consideration ofeffects on functional or antigenic domains of the protein. There are anumber of attachment methods available to those skilled in the art,e.g., EP 0 401 384 herein incorporated by reference (coupling PEG toG-CSF), see also Malik et al., 1992, Exp. Hematol 20:1028-1035(reporting pegylation of GM-CSF using tresyl chloride). For example,polyethylene glycol may be covalently bound through amino acid residuesvia a reactive group, such as, a free amino or carboxyl group. Reactivegroups are those to which an activated polyethylene glycol molecule maybe bound. The amino acid residues having a free amino group includelysine residues and the—terminal amino acid residues; those having afree carboxyl group include aspartic acid residues glutamic acidresidues and the C-terminal amino acid residue. Sulfhydrl groups mayalso be used as a reactive group for attaching the polyethylene glycolmolecule(s). Preferred for therapeutic purposes is attachment at anamino group, such as attachment at the N-terminus or lysine group.

Nucleic Acids

[0141] In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I- III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

[0142] Mutations can be made in a nucleic acid encoding the polypeptideof the present invention such that a particular codon is changed to acodon which codes for a different amino acid. Such a mutation isgenerally made by making the fewest nucleotide changes possible. Asubstitution mutation of this sort can be made to change an amino acidin the resulting protein in a non-conservative manner (i.e., by changingthe codon from an amino acid belonging to a grouping of amino acidshaving a particular size or characteristic to an amino acid belonging toanother grouping) or in a conservative manner (i.e., by changing thecodon from an amino acid belonging to a grouping of amino acids having aparticular size or characteristic to an amino acid belonging to the samegrouping). Such a conservative change generally leads to less change inthe structure and function of the resulting protein. A non-conservativechange is more likely to alter the structure, activity or function ofthe resulting protein. The present invention should be considered toinclude sequences containing conservative changes which do notsignificantly alter the activity or binding characteristics of theresulting protein. Substitutes for an amino acid within the sequence maybe selected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. Amino acids containing aromatic ring structures arephenylalanine, tryptophan, and tyrosine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Such alterations will not beexpected to affect apparent molecular weight as determined bypolyacrylamide gel electrophoresis, or isoelectric point.

[0143] Particularly preferred substitutions are:

[0144] Lys for Arg and vice versa such that a positive charge may bemaintained;

[0145] Glu for Asp and vice versa such that a negative charge may bemaintained;

[0146] Ser for Thr such that a free —OH can be maintained; and

[0147] Gln for Asn such that a free NH₂ can be maintained.

[0148] Synthetic DNA sequences allow convenient construction of geneswhich will express analogs or “muteins”. A general method forsite-specific incorporation of unnatural amino acids into proteins isdescribed inNoren, et al. Science, 244:182-188 (April 1989). This methodmay be used to create analogs with unnatural amino acids.

[0149] This invention provides an isolated nucleic acid encoding apolypeptide comprising an amino acid sequence of a streptococcal Emapolypeptide. This invention provides an isolated nucleic acid encoding apolypeptide comprising an amino acid sequence of a streptococcal Emapolypeptide. This invention provides an isolated nucleic acid encoding apolypeptide comprising an amino acid sequence of a Group B streptococcalEma polypeptide selected from the group of EmaA, EmaB, EmaC, EmaD andEmaE. This invention provides an isolated nucleic acid encoding apolypeptide comprising an amino acid sequence of a Group B streptococcalEma protein selected from the group of Ema proteins EmA, EmaB, EmaC.EmaD and EmaE as set forth in FIGS. 2-6. The invention provides anisolated nucleic acid encoding a polypeptide comprising an amino acidsequence of a bacterial Ema polypeptide selected from the group of SEQID NO: 23, 26, 29, 32 and 37. In particular embodiments the nucleic acidis set forth in any of SEQ ID NOS: 1, 3, 5, 7, 9, 24, 27, 30, and 33,including fragments, mutants, variants, analogs, or derivatives,thereof. The nucleic acid is DNA, cDNA, genomic DNA, RNA Further, theisolated nucleic acid may be operatively linked to a promoter of RNAtranscription.

[0150] The present invention also relates to isolated nucleic acids,such as recombinant DNA molecules or cloned genes, or degeneratevariants thereof mutants, analogs, or fragments thereof, which encodethe isolated polypeptide or which competitively inhibit the activity ofthe polypeptide. The present invention further relates to isolatednucleic acids, such as recombinant DNA molecules or cloned genes, ordegenerate variants thereof, mutants, analogs, or fragments thereof,which encode a GBS Ema polypeptide, particularly selected from the groupof EmaA, EmaB, EmaC, EmaD and EmaE. Preferably, the isolated nucleicacid, which includes degenerates, variants, mutants, analogs, orfragments thereof, has a sequence as set forth in SEQ ID NOS: 1, 3, 5, 7or 9. In a further embodiment of the invention, the DNA sequence of therecombinant DNA molecule or cloned gene may be operatively linked to anexpression control sequence which may be introduced into an appropriatehost. The invention accordingly extends to unicellular hosts transformedwith the cloned gene or recombinant DNA molecule comprising a DNAsequence encoding an Ema protein, particularly selected from the groupof EmaA, EmaB, EmaC, EmaD and EmaE, and more particularly, the DNAsequences or fragments thereof determined from the sequences set forthabove.

[0151] In a particular embodiment, the nucleic acid encoding the EmaApolypeptide has the sequence selected from the group comprising SEQ IDNO: 1; a sequence that hybridizes to SEQ ID NO: 1 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:1 or a sequence thathybridizes to SEQ ID NO: 1 under moderate stringency hybridizationconditions; degenerate variants thereof; alleles thereof; andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaA polypeptide has the sequence selected from thegroup comprising SEQ ID NO: 1; a sequence complementary to SEQ ID NO: 1;or a homologous sequence which is substantially similar to SEQ ID NO: 1.In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO: 1.

[0152] In a particular embodiment, the nucleic acid encoding the EmaBpolypeptide has the sequence selected from the group comprising SEQ IDNO:3; a sequence that hybridizes to SEQ ID NO:3 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:3 or a sequence thathybridizes to SEQ ID NO:3 under moderate stringency hybridizationconditions; degenerate variants thereof; alleles thereof; andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaB polypeptide has the sequence selected from thegroup comprising SEQ ID NO:3; a sequence complementary to SEQ ID NO:3;or a homologous sequence which is substantially similar to SEQ ID NO:3.In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:3.

[0153] In a particular embodiment, the nucleic acid encoding the EmaCpolypeptide has the sequence selected from the group comprising SEQ IDNO:5; a sequence that hybridizes to SEQ ID NO:5 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:5 or a sequence thathybridizes to SEQ ID NO:5 under moderate stringency hybridizationconditions; degenerate variants thereof; alleles thereof; andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaC polypeptide has the sequence selected from thegroup comprising SEQ ID NO:5; a sequence complementary to SEQ ID NO:5;or a homologous sequence which is substantially similar to SEQ ID NO:5.In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:5.

[0154] In a particular embodiment, the nucleic acid encoding the EmaDpolypeptide has the sequence selected from the group comprising SEQ IDNO:7; a sequence that hybridizes to SEQ ID NO:7 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:7 or a sequence thathybridizes to SEQ ID NO:7 under moderate stringency hybridizationconditions; degenerate variants thereof; alleles thereof; andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaD polypeptide has the sequence selected from thegroup comprising SEQ ID NO:7; a sequence complementary to SEQ ID NO:7;or a homologous sequence which is substantially similar to SEQ ID NO:7.In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:7.

[0155] In a particular embodiment, the nucleic acid encoding the EmaEpolypeptide has the sequence selected from the group comprising SEQ IDNO:9; a sequence that hybridizes to SEQ ID NO:9 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:9 or a sequence thathybridizes to SEQ ID NO:9 under moderate stringency hybridizationconditions; degenerate variants thereof; alleles thereof; andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaE polypeptide has the sequence selected from thegroup comprising SEQ ID NO:9; a sequence complementary to SEQ ID NO:9;or a homologous sequence which is substantially similar to SEQ ID NO:9In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:9.

[0156] A nucleic acid capable of encoding a GBS polypeptide EmaA, EmaB,EmaC, EmaD or EmaE which is a recombinant DNA molecule is furtherprovided. Such a recombinant DNA molecule wherein the DNA molecule isoperatively linked to an expression control sequence is also providedherein.

[0157] The present invention relates to nucleic acid vaccines or DNAvaccines comprising nucleic acids encoding immunogenic bacterial Emapolypeptides, particularly immunogenic streptococcal Ema polypeptides.The present invention relates to nucleic acid vaccines or DNA vaccinescomprising nucleic acids encoding immunogenic GBS Ema polypeptides,particularly selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE.The present invention relates to nucleic acid vaccines or DNA vaccinescomprising nucleic acids encoding one or more immunogenic GBS Emapolypeptide or a fragment thereof or any combination of one or more Emapolypeptide EmaA, EmaB, EmaC, EmaD or EmaE with at least one other GBSpolypeptide, particularly wherein said other GBS polypeptide is selectedfrom the group of Spb1, Spb2, C protein alpha antigen, Rib andimmunogenic polypeptide fragments thereof.

[0158] The invention further relates to a vaccine for protection of ananimal subject from infection with a streptococcal bacterium comprisinga vector containing a gene encoding an Ema polypeptide, particularlyselected from the group of EmaA, EmaB, EmaC, EmaD and EmaE, operativelyassociated with a promoter capable of directing expression of the genein the subject. The invention further relates to a vaccine forprotection of an animal subject from infection with a Group Bstreptococcal bacterium comprising a vector containing a gene encodingan Ema polypeptide selected from the group of EmaA, EmaB, EmaC, EmaD andEmaE operatively associated with a promoter capable of directingexpression of the gene in the subject. The present invention furtherprovides a nucleic acid vaccine comprising a recombinant DNA moleculecapable of encoding a GBS polypeptide EmaA, EmaB, EmaC, EmaD or EmaE.

[0159] The present invention provides a vector which comprises thenucleic acid capable of encoding encoding a bacterial Ema polypeptide,particularly a streptococcal Ema polypeptide. The present inventionprovides a vector which comprises the nucleic acid capable of encodingencoding an Ema polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE and a promoter. The present invention provides a vectorwhich comprises the nucleic acid of any of SEQ ID NO: 1, 3, 5, 7, 9, 24,27, 30, and 33, and a promoter. The invention contemplates a vectorwherein the promoter comprises a bacterial, yeast, insect or mammalianpromoter. The invention contemplates a vector wherein the vector is aplasmid, cosmid, yeast artificial chromosome (YAC), bacteriophage oreukaryotic viral DNA.

[0160] The present invention further provides a host vector system forthe production of a polypeptide which comprises the vector capable ofencoding encoding an Ema polypeptide, particularly selected from thegroup of EmaA, EmaB, EmaC, EmaD and EmaE, in a suitable host cell. Ahost vector system is provided wherein the suitable host cell comprisesa prokaryotic or eukaryotic cell. A unicellular host transformed with arecombinant DNA molecule or vector capable of encoding encoding an Emapolypeptide, particularly selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE, is thereby provided.

[0161] A “vector” is a replicon, such as plasmid, phage or cosmid, towhich another DNA segment may be attached so as to bring about thereplication of the attached segment.

[0162] A “DNA” or “DNA molecule” refers to the polymeric form ofdeoxyribonucleotides (adenine, guanine, thymine, or cytosine) in itseither single stranded form, or a double-stranded helix. This termrefers only to the primary and secondary structure of the molecule, anddoes not limit it to any particular tertiary forms. Thus, this termincludes double-stranded DNA found, inter alia, in linear DNA molecules(e.g., restriction fragments), viruses, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA).

[0163] An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

[0164] A DNA “coding sequence” is a double-stranded DNA sequence whichis transcribed and translated into a polypeptide in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence in the case of eukaryoticmRNA.

[0165] Transcriptional and translational control sequences are DNAregulatory sequences, such as promoters, enhancers, polyadenylationsignals, terminators, and the like, that provide for the expression of acoding sequence in a host cell.

[0166] A “promoter sequence” is a DNA regulatory region capable ofbinding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. For purposes of defining thepresent invention, the promoter sequence is bounded at its 3′ terminusby the transcription initiation site and extends upstream (5′ direction)to include the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the −10 and −35 consensus sequences.

[0167] An “expression control sequence” is a DNA sequence that controlsand regulates the transcription and translation of another DNA sequence.A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

[0168] A “signal sequence” can be included before the coding sequence.This sequence encodes a signal peptide, N-terminal to the polypeptide,that communicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

[0169] The term “oligonucleotide,” as used herein in referring to theprobe of the present invention, is defined as a molecule comprised oftwo or more ribonucleotides, preferably more than three. Its exact sizewill depend upon many factors which, in turn, depend upon the ultimatefunction and use of the oligonucleotide.

[0170] The term “primer” as used herein refers to an oligonucleotide,whether occurring naturally as in a purified restriction digest orproduced synthetically, which is capable of acting as a point ofinitiation of synthesis when placed under conditions in which synthesisof a primer extension product, which is complementary to a nucleic acidstrand, is induced, i.e., in the presence of nucleotides and an inducingagent such as a DNA polymerase and at a suitable temperature and pH. Theprimer may be either single-stranded or double-stranded and must besufficiently long to prime the synthesis of the desired extensionproduct in the presence of the inducing agent. The exact length of theprimer will depend upon many factors, including temperature, source ofprimer and use of the method. For example, for diagnostic applications,depending on the complexity of the target sequence, the oligonucleotideprimer typically contains 15-25 or more nucleotides, although it maycontain fewer nucleotides.

[0171] The primers herein are selected to be “substantially”complementary to different strands of a particular target DNA sequence.This means that the primers must be sufficiently complementary tohybridize with their respective strands. Therefore, the primer sequenceneed not reflect the exact sequence of the template. For example, anon-complementary nucleotide fragment may be attached to the 5′ end ofthe primer, with the remainder of the primer sequence beingcomplementary to the strand. Alternatively, non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementarity with the sequence of thestrand to hybridize therewith and thereby form the template for thesynthesis of the extension product.

[0172] As used herein, the terms “restriction endonucleases” and“restriction enzymes” refer to bacterial enzymes, each of which cutdouble-stranded DNA at or near a specific nucleotide sequence.

[0173] A cell has been “transformed” by exogenous or heterologous DNAwhen such DNA has been introduced inside the cell. The transforming DNAmay or may not be integrated (covalently linked) into chromosomal DNAmaking up the genome of the cell. In prokaryotes, yeast, and mammaliancells for example, the transforming DNA may be maintained on an episomalelement such as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

[0174] Two DNA sequences are “substantially homologous” when at leastabout 75% (preferably at least about 80%, and most preferably at leastabout 90 or 95%) of the nucleotides match over the defined length of theDNA sequences. Sequences that are substantially homologous can beidentified by comparing the sequences using standard software availablein sequence data banks, or in a Southern hybridization experiment under,for example, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

[0175] A DNA sequence is “operatively linked” to an expression controlsequence when the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted in front of the gene.

[0176] The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65° C. forboth hybridization and wash. However, one skilled in the art willappreciate that such “standard hybridization conditions” are dependenton particular conditions including the concentration of sodium andmagnesium in the buffer, nucleotide sequence length and concentration,percent mismatch, percent formamide, and the like. Also important in thedetermination of “standard hybridization conditions” is whether the twosequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standardhybridization conditions are easily determined by one skilled in the artaccording to well known formulae, wherein hybridization is typically10-20° C. below the predicted or determined T_(m) with washes of higherstringency, if desired.

[0177] It should be appreciated that also within the scope of thepresent invention are DNA sequences encoding an Ema polypeptide EmaA,EmaB, EmaC, EmaD or EmaE which code for an Ema polypeptide having thesame amino acid sequence as any of SEQ ID NOS:2, 4, 6, 8 or 10, butwhich are degenerate to any of SEQ ID NOS:1, 3, 5, 7 or 9. By“degenerate to” is meant that a different three-letter codon is used tospecify a particular amino acid. It is well known in the art that thefollowing codons can be used interchangeably to code for each specificamino acid: Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L) UUAor UUG or GUU or CUC or CUA or GUG Isoleucine (Ile or I) AUU or AUC orAUA Methionine (Met or M) AUG Valine (Val or V) GUU or GUC of GUA or GUGSerine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro orP) CCU or CCC or CCA or CCG Threonine (Thr or T) ACU or ACC or ACA orACG Alanine (Ala or A) GCU or GCG or GCA or GCG Tyrosine (Tyr or Y) UAUor UAC Histidine (His or H) CAU or CAC Glutamine (Gln or Q) CAA or CAGAsparagine (Asn or N) AAU or AAC Lysine (Lys or K) AAA or AAG AsparticAcid (Asp or D) GAU or GAC Glutamic Acid (Glu or E) GAA or GAG Cysteine(Cys or C) UGU or UGC Arginine (Arg or R) CGU or CGC or CGA or CGG orAGA or AGG Glycine (Gly or G) GGU or GGC or GGA or GGG Tryptophan (Trpor W) UGG Termination codon UAA (ochre) or UAG (amber) or UGA (opal)

[0178] It should be understood that the codons specified above are forRNA sequences. The corresponding codons for DNA have a T substituted forU.

[0179] Mutations can be made in SEQ ID NOS: 1, 3, 5, 7 or 9 such that aparticular codon is changed to a codon which codes for a different aminoacid. Such a mutation is generally made by making the fewest nucleotidechanges possible. A substitution mutation of this sort can be made tochange an amino acid in the resulting protein in a non-conservativemanner (i.e., by changing the codon from an amino acid belonging to agrouping of amino acids having a particular size or characteristic to anamino acid belonging to another grouping) or in a conservative manner(i.e., by changing the codon from an amino acid belonging to a groupingof amino acids having a particular size or characteristic to an aminoacid belonging to the same grouping). Such a conservative changegenerally leads to less change in the structure and function of theresulting protein. A non-conservative change is more likely to alter thestructure, activity or function of the resulting protein. The presentinvention should be considered to include sequences containingconservative changes which do not significantly alter the activity orbinding characteristics of the resulting protein.

[0180] Two amino acid sequences are “substantially homologous” when atleast about 70% of the amino acid residues (preferably at least about80%, and most preferably at least about 90 or 95%) are identical, orrepresent conservative substitutions.

[0181] A “heterologous” region of the DNA construct is an identifiablesegment of DNA within a larger DNA molecule that is not found inassociation with the larger molecule in nature. Thus, when theheterologous region encodes a mammalian gene, the gene will usually beflanked by DNA that does not flank the mammalian genomic DNA in thegenome of the source organism. Another example of a heterologous codingsequence is a construct where the coding sequence itself is not found innature (e.g., a cDNA where the genomic coding sequence contains introns,or synthetic sequences having codons different than the native gene).Allelic variations or naturally-occurring mutational events do not giverise to a heterologous region of DNA as defined herein.

[0182] A DNA sequence is “operatively linked” to an expression controlsequence when the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted in front of the gene.

[0183] Further this invention also provides a vector which comprises theabove-described nucleic acid molecule. The promoter may be, or isidentical to, a bacterial, yeast, insect or mammalian promoter. Further,the vector may be a plasmid, cosmid, yeast artificial chromosome (YAC),bacteriophage or eukaryotic viral DNA. Other numerous vector backbonesknown in the art as useful for expressing protein may be employed. Suchvectors include, but are not limited to: adenovirus, simian virus 40(SV40), cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), Moloneymurine leukemia virus, DNA delivery systems, i.e. liposomes, andexpression plasmid delivery systems. Such vectors may be obtainedcommercially or assembled from the sequences described by methodswell-known in the art.

[0184] This invention also provides a host vector system for theproduction of a polypeptide which comprises the vector of a suitablehost cell. A wide variety of unicellular host cells are also useful inexpressing the DNA sequences of this invention. These hosts may includewell known eukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, such as CHO, R1.1, B-W and L-M cells, African Green Monkey kidneycells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g.,Sf9), and human cells and plant cells in tissue culture.

[0185] A wide variety of host/expression vector combinations may beemployed in expressing the DNA sequences of this invention. Usefulexpression vectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol E1, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4;phage DNAs, e.g., the numerous derivatives of phage λ, M13 andfilamentous single stranded phage DNA; yeast plasmids such as the 2μplasmid or derivatives thereof; vectors useful in eukaryotic cells, suchas vectors useful in insect or mammalian cells; vectors derived fromcombinations of plasmids and phage DNAs, such as plasmids that have beenmodified to employ phage DNA or other expression control sequences; andthe like.

[0186] Any of a wide variety of expression control sequences—sequencesthat control the expression of a DNA sequence operatively linked toit—may be used in these vectors to express the DNA sequences of thisinvention. Such useful expression control sequences include, forexample, the early or late promoters of SV40, CMV, vaccinia, polyoma oradenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the LTR system, the major operator and promoter regions of phageλ, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase (e.g., Pho5), the promoters of the yeast α-matingfactors, and other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof.

[0187] It will be understood that not all vectors, expression controlsequences and hosts will function equally well to express the DNAsequences of this invention. Neither will all hosts function equallywell with the same expression system. However, one skilled in the artwill be able to select the proper vectors, expression control sequences,and hosts without undue experimentation to accomplish the desiredexpression without departing from the scope of this invention. Forexample, in selecting a vector, the host must be considered because thevector must function in it. The vector's copy number, the ability tocontrol that copy number, and the expression of any other proteinsencoded by the vector, such as antibiotic markers, will also beconsidered.

[0188] In selecting an expression control sequence, a variety of factorswill normally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

[0189] This invention further provides a method of producing apolypeptide which comprises growing the above-described host vectorsystem under suitable conditions permitting the production of thepolypeptide and recovering the polypeptide so produced.

[0190] As used herein, “pg” means picogram, “ng” means nanogram, “ug” or“μg” mean microgram, “mg” means milligram, “ul” or “μl” mean microliter,“ml” means milliliter, “l” means liter.

[0191] The present invention extends to the preparation of antisenseoligonucleotides and ribozymes that may be used to interfere with theexpression of one or more Ema protein at the translational level. Thisapproach utilizes antisense nucleic acid and ribozymes to blocktranslation of a specific mRNA, either by masking that mRNA with anantisense nucleic acid or cleaving it with a ribozyme.

[0192] Antisense nucleic acids are DNA or RNA molecules that arecomplementary to at least a portion of a specific mRNA molecule. (SeeWeintraub, 1990; Marcus-Sekura, 1988.) In the cell, they hybridize tothat mRNA, forming a double stranded molecule. The cell does nottranslate an mRNA in this double-stranded form. Therefore, antisensenucleic acids interfere with the expression of mRNA into protein.Oligomers of about fifteen nucleotides and molecules that hybridize tothe AUG initiation codon will be particularly efficient, since they areeasy to synthesize and are likely to pose fewer problems than largermolecules when introducing them into Ema-producing cells. Antisensemethods have been used to inhibit the expression of many genes in vitro(Marcus-Sekura, 1988; Hambor et al., 1988).

[0193] Ribozymes are RNA molecules possessing the ability tospecifically cleave other single stranded RNA molecules in a mannersomewhat analogous to DNA restriction endonucleases. Ribozymes werediscovered from the observation that certain mRNAs have the ability toexcise their own introns. By modifying the nucleotide sequence of theseRNAs, researchers have been able to engineer molecules that recognizespecific nucleotide sequences in an RNA molecule and cleave it (Cech,1988.). Because they are sequence-specific, only mRNAs with particularsequences are inactivated.

[0194] Investigators have identified two types of ribozymes,Tetrahymena-type and “hammerhead”-type. (Hasselhoff and Gerlach, 1988)Tetrahymena-type ribozymes recognize four-base sequences, while“hammerhead”-type recognize eleven- to eighteen-base sequences. Thelonger the recognition sequence, the more likely it is to occurexclusively in the target mRNA species. Therefore, hammerhead-typeribozymes are preferable to Tetrahymena-type ribozymes for inactivatinga specific mRNA species, and eighteen base recognition sequences arepreferable to shorter recognition sequences.

Antibodies

[0195] This invention further provides an antibody capable ofspecifically recognizing or binding to the isolated Ema polypeptide ofthe present invention. The antibody may be a monoclonal or polyclonalantibody. Further, the antibody may be labeled with a detectable markerthat is either a radioactive, calorimetric, fluorescent, or aluminescent marker. The labeled antibody may be a polyclonal ormonoclonal antibody. In one embodiment, the labeled antibody is apurified labeled antibody. Methods of labeling antibodies are well knownin the art.

[0196] In a further aspect, the present invention provides a purifiedantibody to a bacterial Ema polypeptide, particularly a streptococcalEma polypeptide. In a still further aspect, the present inventionprovides a purified antibody to a Group B sreptococcal polypeptideselected from the group of EmaA, EmaB, EmaC, EmaD and EmaE.

[0197] Antibodies against the isolated polypeptides of the presentinvention include naturally raised and recombinantly preparedantibodies. These may include both polyclonal and monoclonal antibodiesprepared by known genetic techniques, as well as bi-specific (chimeric)antibodies, and antibodies including other functionalities suiting themfor diagnostic use. Such antibodies can be used in immunoassays todiagnose infection with a particular strain or species of bacteria. Theantibodies can also be used for passive immunization to treat aninfection with Group B streptococcal bacteria. These antibodies may alsobe suitable for modulating bacterial adherence and/or invasion includingbut not limited to acting as competitive agents.

[0198] The present invention provides a monoclonal antibody to a Group Bstreptococcal poypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE. The invention thereby extends to an immortal cell linethat produces a monoclonal antibody to a Group B streptococcalpoypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE.

[0199] An antibody to an Ema polypeptide, particularly selected fromEmaA, EmaB, EmaC. EmaD or EmaE, labeled with a detectable label isfurther provided. In particular embodiments, the label may selected fromthe group consisting of an enzyme, a chemical which fluoresces, and aradioactive element.

[0200] The term “antibody” includes, by way of example, both naturallyoccurring and non-naturally occurring antibodies. Specifically, the term“antibody” includes polyclonal and monoclonal antibodies, and fragmentsthereof. Furthermore, the term “antibody” includes chimeric antibodiesand wholly synthetic antibodies, and fragments thereof. Such antibodiesinclude but are not limited to polyclonal, monoclonal, chimeric, singlechain, Fab fragments, and an Fab expression library.

[0201] An “antibody” is any immunoglobulin, including antibodies andfragments thereof, that binds a specific epitope. The term encompassespolyclonal, monoclonal, and chimeric antibodies, the last mentioneddescribed in further detail in U.S. Pat. Nos. 4,816,397 and 4,816,567.

[0202] An “antibody combining site” is that structural portion of anantibody molecule comprised of heavy and light chain variable andhypervariable regions that specifically binds antigen.

[0203] The phrase “antibody molecule” in its various grammatical formsas used herein contemplates both an intact immunoglobulin molecule andan immunologically active portion of an immunoglobulin molecule.

[0204] Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contains the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v), which portionsare preferred for use in the therapeutic methods described herein.

[0205] Fab and F(ab′)₂ portions of antibody molecules are prepared bythe proteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibody molecules by methods that are well-known.See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab′antibody molecule portions are also well-known and are produced fromF(ab′)₂ portions followed by reduction of the disulfide bonds linkingthe two heavy chain portions as with mercaptoethanol, and followed byalkylation of the resulting protein mercaptan with a reagent such asiodoacetamide. An antibody containing intact antibody molecules ispreferred herein.

[0206] The phrase “monoclonal antibody” in its various grammatical formsrefers to an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

[0207] Various procedures known in the art may be used for theproduction of polyclonal antibodies to polypeptide or derivatives oranalogs thereof (see, e.g., Antibodies—A Laboratory Manual, Harlow andLane, eds., Cold Spring Harbor Laboratory Press: Cold Spring Harbor,N.Y., 1988). For the production of antibody, various host animals can beimmunized by injection with the Group B streptococcal Ema polypeptide,an immunogenic fragment thereof, or a derivative (e.g., fragment orfusion protein) thereof, including but not limited to rabbits, mice,rats, sheep, goats, etc. In one embodiment, the polypeptide can beconjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA)or keyhole limpet hemocyanin (KLH). Various adjuvant may be used toincrease the immunological response, depending on the host species.

[0208] For preparation of monoclonal antibodies, or fragment, analog, orderivative thereof, any technique that provides for the production ofantibody molecules by continuous cell lines in culture may be used (see,e.g., Antibodies—A Laboratory Manual, Harlow and Lane, eds., Cold SpringHarbor Laboratory Press: Cold Spring Harbor, N.Y., 1988). These includebut are not limited to the hybridoma technique originally developed byKohler and Milstein (1975, Nature 256:495-497), as well as the triomatechnique, the human B-cell hybridoma technique (Kozbor et al., 1983,Immunology Today 4:72), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96). Monoclonal antibodiescan be produced in germ-free animals utilizing recent technology(PCT/US90/02545). Human antibodies may be used and can be obtained byusing human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A80:2026-2030) or by transforming human B cells with EBV virus in vitro(Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, pp. 77-96). In fact, according to the invention, techniquesdeveloped for the production of “chimeric antibodies” (Morrison et al.,1984, J. Bacteriol. 159-870; Neuberger et al., 1984, Nature 312:604-608;Takeda et al., 1985, Nature 314:452-454) by splicing the genes from amouse antibody molecule specific for a polypeptide together with genesfrom a human antibody molecule of appropriate biological activity can beused; such antibodies are within the scope of this invention. Such humanor humanized chimeric antibodies are preferred for use in therapy ofhuman infections or diseases, since the human or humanized antibodiesare much less likely than xenogenic antibodies to induce an immuneresponse, in particular an allergic response, themselves. An additionalembodiment of the invention utilizes the techniques described for theconstruction of Fab expression libraries (Huse et al, 1989, Science246:1275-1281) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity for the polypeptide, or itsderivatives, or analogs.

[0209] Antibody fragments which contain the idiotype of the antibodymolecule can be generated by known techniques. For example, suchfragments include but are not limited to: the F(ab′)₂ fragment which canbe produced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

[0210] In the production of antibodies, screening for the desiredantibody can be accomplished by techniques known in the art, e.g.,radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitinreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

[0211] Antibodies can be labeled for detection in vitro, e.g., withlabels such as enzymes, fluorophores, chromophores, radioisotopes, dyes,colloidal gold, latex particles, and chemiluminescent agents.Alternatively, the antibodies can be labeled for detection in vivo,e.g., with radioisotopes (preferably technetium or iodine); magneticresonance shift reagents (such as gadolinium and manganese); orradio-opaque reagents.

[0212] The labels most commonly employed for these studies areradioactive elements, enzymes, chemicals which fluoresce when exposed toultraviolet light, and others. A number of fluorescent materials areknown and can be utilized as labels. These include, for example,fluorescein, rhodamine, auramine, Texas Red, AMCA blue and LuciferYellow. A particular detecting material is anti-rabbit antibody preparedin goats and conjugated with fluorescein through an isothiocyanate. Thepolypeptide can also be labeled with a radioactive element or with anenzyme. The radioactive label can be detected by any of the currentlyavailable counting procedures. The preferred isotope may be selectedfrom ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I,and ¹⁸⁶Re.

[0213] Enzyme labels are likewise useful, and can be detected by any ofthe presently utilized calorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle-by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be utilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090;3,850,752; and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

Diagnostic Applications

[0214] The present invention also relates to a variety of diagnosticapplications, including methods for identifying or monitoringstreptococcal infections. The present invention also relates to avariety of diagnostic applications, including methods for identifying ormonitoring Group B streptococcal infections. The present inventionfurther relates to diagnostic applications or methods utilizing thepolypeptides of the present invention, immunogenically recognizedfragments thereof, or antibodies thereto. Such methods include theanalysis and evaluation of agents, analogs or compounds which modulatethe activity of the Ema polypeptides. The Ema polypeptides may also beutilized in diagnostic methods and assays for monitoring and determiningimmunological response and antibody response upon streptococcalinfection or vaccination.

[0215] As described in detail above, antibody(ies) to the Emapolypeptides or fragments thereof can be produced and isolated bystandard methods including the well known hybridoma techniques. Forconvenience, the antibody(ies) to the Ema polypeptides will be referredto herein as Ab₁ and antibody(ies) raised in another species as Ab₂.

[0216] The presence of streptococci in cells can be ascertained by theusual immunological procedures applicable to such determinations. Anumber of useful procedures are known. Procedures which are especiallyuseful utilize either the Ema polypeptides labeled with a detectablelabel, antibody against the Ema polypeptides labeled with a detectablelabel, or secondary antibody labeled with a detectable label.

[0217] The procedures and their application are all familiar to thoseskilled in the art and accordingly may be utilized within the scope ofthe present invention. The “competitive” procedure, is described in U.S.Pat. Nos. 3,654,090 and 3,850,752. The “sandwich” procedure, isdescribed in U.S. Pat. Nos. RE 31,006 and 4,016,043. Still otherprocedures are known such as the “double antibody,” or “DASP” procedure.

[0218] In each instance, the Ema polypeptides forms complexes with oneor more antibody(ies) or binding partners and one member of the complexis labeled with a detectable label. The fact that a complex has formedand, if desired, the amount thereof, can be determined by known methodsapplicable to the detection of labels.

[0219] In a further embodiment of this invention, commercial test kitssuitable for use by a medical specialist may be prepared to determinethe presence or absence of stretococci, particularly of streptococciexpressing one or more Ema polypeptide selected from the group of EmaA,EmaB, EmaC, EmaD and EmaE. In as much as the ema locus, as describedherein, is found in the genomic DNA of many, if not all, serotypes ofGroup B streptococci, it is a useful general marker for Group Bstreptococci. In as much as Ema homologs exist in other species ofstreptococci, including Group A and S. pneumoniae, it is a usefulgeneral marker for streptococci. Therefore, commercial test kits fordetermining the presence or absence of streptococci, and therebydetermining whether an individual is infected with streptococci arecontemplated and provided by this invention. Therefore, commercial testkits for determining the presence or absence of Group B streptococci,and thereby determining whether an individual is infected with Group Bstreptococci are contemplated and provided by this invention.

[0220] The present invention includes methods for determining andmonitoring infection by streptococci by detecting the presence of astreptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE. In a particular such method, the streptococcal Emapolypeptide is measured by:

[0221] a. contacting a sample in which the presence or activity of aStreptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE is suspected with an antibody to the said streptococcalpolypeptide under conditions that allow binding of the streptococcalpolypeptide to the antibody to occur; and

[0222] b. detecting whether binding has occurred between thestreptococcal polypeptide from the sample and the antibody;

[0223] wherein the detection of binding indicates the presence oractivity of the streptococcal polypeptide in the sample.

[0224] The present invention includes methods for determining andmonitoring infection by Group B streptococci by detecting the presenceof a Group B streptococcal polypeptide selected from the group of EmaA,EmaB, EmaC, EmaD and EmaE. In a particular such method, thestreptococcal Ema polypeptide is measured by:

[0225] a. contacting a sample in which the presence or activity of aGroup B Streptococcal polypeptide selected from the group of EmaA, EmaB,EmaC, EmaD and EmaE is suspected with an antibody to the said Group Bstreptococcal polypeptide under conditions that allow binding of theGroup B streptococcal polypeptide to the antibody to occur; and

[0226] b. detecting whether binding has occurred between the Group Bstreptococcal polypeptide from the sample and the antibody;

[0227] wherein the detection of binding indicates the presence oractivity of the a Group B streptococcal polypeptide in the sample.

[0228] The present invention further provides a method for detecting thepresence of a bacterium having a gene encoding a Group B polypeptideselected from the group of emaA, emaB, emaC, emaD and emaE, comprising:

[0229] a. contacting a sample in which the presence or activity of thebacterium is suspected with an oligonucleotide which hybridizes to aGroup B streptococcal polypeptide gene selected from the group of emaA,emaB, emaC, emaD and emaE, under conditions that allow specifichybridization of the oligonucleotide to the gene to occur; and

[0230] b. detecting whether hybridization has occurred between theoligonucleotide and the gene;

[0231] wherein the detection of hybridization indicates that presence oractivity of the bacterium in the sample.

[0232] The invention includes an assay system for screening of potentialcompounds effective to modulate the activity of a bacterial Ema proteinof the present invention. In one instance, the test compound, or anextract containing the compound, could be administered to a cellularsample expressing the particular Ema protein to determine the compound'seffect upon the activity of the protein by comparison with a control. Ina further instance the test compound, or an extract containing thecompound, could be administered to a cellular sample expressing the Emaprotein to determine the compound's effect upon the activity of theprotein, and thereby on adherence of said cellular sample to host cells,by comparison with a control.

[0233] Accordingly, a test kit may be prepared for the demonstration ofthe presence of Ema polypeptide or Ema activity in cells, comprising:

[0234] (a) a predetermined amount of at least one labeledimmunochemically reactive component obtained by the direct or indirectattachment of the Ema polypeptide or a specific binding partner thereto,to a detectable label;

[0235] (b) other reagents; and

[0236] (c) directions for use of said kit.

[0237] More specifically, the diagnostic test kit may comprise:

[0238] (a) a known amount of the Ema polypeptide as described above (ora binding partner) generally bound to a solid phase to form animmunosorbent, or in the alternative, bound to a suitable tag, or pluralsuch end products, etc. (or their binding partners) one of each;

[0239] (b) if necessary, other reagents; and

[0240] (c) directions for use of said test kit.

[0241] In a further variation, the test kit may be prepared and used forthe purposes stated above, which operates according to a predeterminedprotocol (e.g. “competitive,” “sandwich,” “double antibody,” etc.), andcomprises:

[0242] (a) a labeled component which has been obtained by coupling theEma polypeptide to a detectable label;

[0243] (b) one or more additional immunochernical reagents of which atleast one reagent is a ligand or an immobilized ligand, which ligand isselected from the group consisting of:

[0244] (i) a ligand capable of binding with the labeled component (a);

[0245] (ii) a ligand capable of binding with a binding partner of thelabeled component (a);

[0246] (iii) a ligand capable of binding with at least one of thecomponent(s) to be determined; and

[0247] (iv) a ligand capable of binding with at least one of the bindingpartners of at least one of the component(s) to be determined; and

[0248] (c) directions for the performance of a protocol for thedetection and/or determination of one or more components of animmunochemical reaction between the Ema polypeptide and a specificbinding partner thereto.

[0249] In accordance with the above, an assay system for screeningpotential drugs effective to modulate the activity of the Emapolypeptide may be prepared. The Ema polypeptide may be introduced intoa test system, and the prospective drug may also be introduced into theresulting cell culture, and the culture thereafter examined to observeany changes in the Ema polypeptide activity of the cells, due either tothe addition of the prospective drug alone, or due to the effect ofadded quantities of the known Ema polypeptide.

Therapeutic Applications

[0250] The therapeutic possibilities that are raised by the existence ofthe Group B streptococcal Ema polypeptides EmaA, EmaB, EmaC, EmaD andEmaE derive from the fact that the Ema polypeptides of the presentinvention are found generally in various serotypes of Group Bstreptococci. In addition, broader therapeutic possibilities that areraised by the existence of Ema homologous polypeptides in variousdistinct species of streptococci, including S. pneumoniae and S.pyogenes. In addition Ema homologous polypeptides have been identifiedin E. faecalis and C. diptheriae. Of particular relevance to theirsuitability in vaccine and immunological therapy is that the Ema A,EmaB, and EmaC polypeptides possess N-terminal sequences consistent witha signal peptide, indicating secretion from the bacterial cell and atleast partial extracellular localization. In addition, the EmaA, EmaB,EmaC, EmaD and EmaE polypeptides demonstrate homology to distinctbacterial proteins involved in or implicated in bacterial adhesion andinvasion. Thus, the Ema polypeptides are anticipated to be involved inor required for streptococcal adhesion to and/or invasion of cells,critical for bacterial survival and virulence in the human host.

Modulators of Exracellular Matrix Adhesin Protein

[0251] Thus, in instances where it is desired to reduce or inhibit theeffects resulting from the extracellular matrix adhesin protein Ema ofthe present invention, an appropriate inhibitor of one or more of theEma proteins, particularly EmaA, EmaB, EmaC, EmaD and EmaE could beintroduced to block the activity of one or more Ema protein.

[0252] The present invention contemplates screens for a modulator of anEma polypeptide, in particular modulating adhesion or invasionfacilitated by EmaA, EmaB, EmaC, EmaD or EmaE. In one such embodiment,an expression vector containing the Ema polypeptide of the presentinvention, or a derivative or analog thereof, is placed into a cell inthe presence of at least one agent suspected of exhibiting Emapolypeptide modulator activity. The cell is preferably a bacterial cell,most preferably a streptococcal cell, or a bacterial host cell. Theamount of adhesion or binding activity is determined and any such agentis identified as a modulator when the amount of adhesion or bindingactivity in the presence of such agent is different than in its absence.The vectors may be introduced by any of the methods described above. Ina related embodiment the GBS Ema polypeptide is expressed instreptococci and the step of determining the amount of adhesion orbinding activity is performed by determining the amount of binding tobacterial host cells cells in vitro.

[0253] When the amount of adhesion or binding activity in the presenceof the modulator is greater than in its absence, the modulator isidentified as an agonist or activator of the Ema polypeptide, whereaswhen the amount of adhesion binding activity in the presence of themodulator is less than in its absence, the modulator is identified as anantagonist or inhibitor of the Ema polypeptide. As any person havingskill in the art would recognize, such determinations as these and thosebelow could require some form of statistical analysis, which is wellwithin the skill in the art.

[0254] Natural effectors found in cells expressing Ema polypeptide canbe fractionated and tested using standard effector assays as exemplifiedherein, for example. Thus an agent that is identified can be a naturallyoccurring adhesion or binding modulator. Alternatively, natural productslibraries can be screened using the assays of the present invention forscreening such agents.,

[0255] Another approach uses recombinant bacteriophage to produce largelibraries. Using the “phage method” [Scott and Smith, 1990, Science249:386-390 (1990); Cwirla, et al., Proc. Natl. Acad. Sci., 87:6378-6382(1990); Devlin et al., Science, 249:404-406 (1990)], very largelibraries can be constructed (10⁶-10⁸ chemical entities). Yet anotherapproach uses primarily chemical methods, of which the Geysen method[Geysen et al., Molecular Immunology 23:709-715 (1986); Geysen et al. J.Immunologic Method 102:259-274 (1987)] and the method of Fodor et al.[Science 251:767-773 (1991)] are examples. Furka et al. [14thInternational Congress of Biochemistry, Volume 5, Abstract FR:013(1988); Furka, Int. J. Peptide Protein Res. 37:487493 (1991)], Houghton[U.S. Pat. No. 4,631,211, issued December 1986] and Rutter et al. [U.S.Pat. No. 5,010,175, issued Apr. 23, 1991] describe methods to produce amixture of peptides that can be tested.

[0256] In another aspect, synthetic libraries [Needels et al., Proc.Natl. Acad. Sci. USA 90:10700-4 (1993); Ohlmeyer et al., Proc. Natl.Acad. Sci. USA 90:10922-10926 (1993); Lam et al., International PatentPublication No. WO 92/00252; Kocis et al., International PatentPublication No. WO 9428028, each of which is incorporated herein byreference in its entirety], and the like can be used to screen for suchan agent.

[0257] This invention provides antagonist or blocking agents whichinclude but are not limited to: peptide fragments, mimetic, a nucleicacid molecule, a ribozyme, a polypeptide, a small molecule, acarbohydrate molecule, a monosaccharide, an oligosaccharide or anantibody. Also, agents which competitively block or inhibitstreptococcal bacterium are contemplated by this invention. Thisinvention provides an agent which comprises an inorganic compound, anucleic acid molecule, an oligonucleotide, an organic compound, apeptide, a peptidomimetic compound, or a protein which inhibits thepolypeptide.

Vaccines

[0258] In a further aspect, the present invention extends to vaccinesbased on the Ema proteins described herein. The present inventionprovides a vaccine comprising one or more Group B streptococcalpolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE,and a pharmaceutically acceptable adjuvant. The present inventionprovides a vaccine comprising one or more bacterial Ema polypeptideselected from the group of polypeptides comprising the amino acidsequence set out in any of SEQ ID NO: 23, 26, 29, 32 and 37, and apharmaceutically acceptable adjuvant.

[0259] The present invention further provides a vaccine comprising oneor more Group B streptococcal polypeptide selected from the group ofEmaA, EmaB, EmaC, EmaD and EmaE, further comprising one or moreadditional GBS antigen. The present invention further provides a vaccinecomprising one or more Group B streptococcal polypeptide selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE, further comprising one ormore antigens selected from the group of the polypeptide Spbl or animmunogenic fragment thereof, the polypeptide Spb2 or an immunogenicfragment thereof, C protein alpha antigen or an immunogenic fragmentthereof, Rib or an immunogenic fragment thereof, Lmb or an immunogenicfragment thereof, C5a-ase or an immunogenic fragment thereof, and GroupB streptococcal polysaccharides or oligosaccharides.

[0260] In another aspect, the invention is directed to a vaccine forprotection of an animal subject from infection with streptococcicomprising an immunogenic amount of one or more streptococcal Emapolypeptide, or a derivative or fragment thereof. The Ema polypeptidemay be particularly selected from the group of EmaA, EmaB, EmaC, EmaD orEmaE, or a derivative or fragment thereof. In a further aspect, theinvention is directed to a vaccine for protection of an animal subjectfrom infection with streptococci comprising an immunogenic amount of oneor more Ema polypeptide EmaA, EmaB, EmaC, EmaD or EmaE, or a derivativeor fragment thereof. In a further aspect, the invention is directed to avaccine for protection of an animal subject from infection with GBScomprising an immunogenic amount of one or more Ema polypeptide EmaA,EmaB, EmaC, EmaD or EmaE, or a derivative or fragment thereof. Such avaccine may contain the protein conjugated covalently to a streptococcalor GBS bacterial polysaccharide or oligosaccharide or polysaccharide oroligosaccharide from one or more streptococcal or GBS serotypes.

[0261] This invention provides a vaccine which comprises a polypeptidebacterial Ema protein and a pharmaceutically acceptable adjuvant orcarrier. In particular, a vaccine is provided which comprises one ormore Ema polypeptides selected from the group of EmaA, EmaB, EmaC, EmaDand EmaE. This invention provides a vaccine which comprises acombination of at least one bacterial Ema protein selected from thegroup of EmaA, EmaB, EmaC, EmaD and EmaE and at least one other Group Bstreptococcal protein particularly Spb1 and/or Spb2 and/or C proteinalpha antigen, and a pharmaceutically acceptable adjuvant or carrier.The Ema polypeptide may comprise an amino acid sequence of a Ema proteinEmaA, EmaB, EmaC, EmaD, EmaE as set forth in FIGS. 2-6 and SEQ ID NOS:2, 4, 6, 8 and 10.

[0262] This invention further provides a vaccine comprising an isolatednucleic acid encoding a bacterial Ema polypeptide and a pharmaceuticallyacceptable adjuvant or carrier. This invention further provides avaccine comprising an isolated nucleic acid encoding a streptococcal Emapolypeptide and a pharmaceutically acceptable adjuvant or carrier. Thisinvention further provides a vaccine comprising an isolated nucleic acidencoding a GBS Ema polypeptide and a pharmaceutically acceptableadjuvant or carrier. This invention further provides a vaccinecomprising isolated nucleic acid encoding one or more GBS Emapolypeptide, particularly selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE and a pharmaceutically acceptable adjuvant or carrier. Thenucleic acid may comprise a nucleic acid sequence of a GBS Emapolypeptide as set forth in any of SEQ ID NOS:1, 3, 5, 7, or 9.

[0263] Active immunity against streptococci can be induced byimmunization (vaccination) with an immunogenic amount of thepolypeptide, or peptide derivative or fragment thereof, and an adjuvant,wherein the polypeptide, or antigenic derivative or fragment thereof, isthe antigenic component of the vaccine. The polypeptide, or antigenicderivative or fragment thereof, may be one antigenic component, in thepresence of other antigenic components in a vaccine. For instance, thepolypeptide of the present invention may be combined with other knownstreptococcal polypeptides or poly/oligosaccharides, or immunogenicfragments thereof, including for instance GB S capsular polysaccharide,Spb1, Spb2, C protein alpha antigen, Rib, Lmb, and C5a-ase in amulti-component vaccine. Such multi-component vaccine may be utilized toenhance immune response, even in cases where the polypeptide of thepresent invention elicits a response on its own. The polypeptide of thepresent invention may also be combined with existing vaccines, wholebacterial or capsule-based vaccines, alone or in combination with otherGBS polypeptides, particularly Spb1 and/or Spb2 and/or C protein alphaantigen and/or Rib to enhance such existing vaccines.

[0264] The term “adjuvant” refers to a compound or mixture that enhancesthe immune response to an antigen. An adjuvant can serve as a tissuedepot that slowly releases the antigen and also as a lymphoid systemactivator that non-specifically enhances the immune response (Hood etal., Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park,Calif., p. 384). Often, a primary challenge with an antigen alone, inthe absence of an adjuvant, will fail to elicit a humoral or cellularimmune response. Adjuvant include, but are not limited to, completeFreund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbonemulsions, keyhole limpet hemocyanins, dinitrophenol, and potentiallyuseful human adjuvant such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum. Preferably, the adjuvant is pharmaceuticallyacceptable.

[0265] The invention further provides a vaccine which comprises anon-adherent, non-virulent mutant, including but not limited to the ema⁻mutants herein described and contemplated. Medaglini et al (Madaglini etal (1995) Proc Natl Acad Sci USA 92;6868-6872) and Oggioni and Pozzi(Oggioni, M. R. and Pozzi, G. (1996) Gene 169:85-90) have previouslydescribed the use of Streptococcus gordonii, a commensal bacterium ofthe human oral cavity, as live vaccine delivery vehicles and forheterologous gene expression. Such ema⁻ mutant can therefore be utilizedas a vehicle for expression of immunogenic proteins for the purposes ofeliciting an immune response to such other proteins in the context ofvaccines. Active immunity against Group B streptococci, can be inducedby immunization (vaccination) with an immunogenic amount of the ema⁻vehicle expressing an immunogenic protein. Also contemplated by thepresent invention is the use of any such ema⁻ mutant in expressing atherapeutic protein in the host in the context of other forms oftherapy.

[0266] The polypeptide of the present invention, or fragments thereof,can be prepared in an admixture with an adjuvant to prepare a vaccine.Preferably, the polypeptide or peptide derivative or fragment thereof,used as the antigenic component of the vaccine is an antigen common toall or many serotypes of GBS bacteria, or common to closely relatedspecies of bacteria, for instance Streptococcus.

[0267] Vectors containing the nucleic acid-based vaccine of theinvention can be introduced into the desired host by methods known inthe art, e.g., transfection, electroporation, micro injection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem.267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut etal., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

[0268] The modes of administration of the vaccine or compositions of thepresent invention may comprise the use of any suitable means and/ormethods for delivering the vaccine or composition to the host animalwhereby they are immumostimulatively effective. Delivery modes mayinclude, without limitation, parenteral administration methods, such asparacancerally, transmucosally, transdermally, intramuscularly,intravenously, intradermally, subcutaneously, intraperitonealy,intraventricularly, intracranially and intratumorally. Preferably, sincethe desired result of vaccination is to elucidate an immune response tothe antigen, and thereby to the pathogenic organism, administrationdirectly, or by targeting or choice of a viral vector, indirectly, tolymphoid tissues, e.g., lymph nodes or spleen, is desirable. Sinceimmune cells are continually replicating, they are ideal target forretroviral vector-based nucleic acid vaccines, since retrovirusesrequire replicating cells. These vaccines and compositions can be usedto immunize mammals, for example, by the intramuscular or parenteralroutes, or by delivery to mucosal surfaces using microparticles,capsules, liposomes and targeting molecules, such as toxins andantibodies. The vaccines and immunogenic compositions may beadministered to mucosal surfaces by, for example, the nasal or oral(intragastric) routes. Alternatively, other modes of administrationincluding suppositories may be desirable. For suppositories, binders andcarriers may include, for example, polyalkylene glycols andtriglycerides. Oral formulations may include normally employedincipients, such as pharmaceutical grades of saccharine, cellulose andmagnesium carbonate.

[0269] These compositions may take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders andcontain 1 to 95% of the immunogenic compositions of the presentinvention. The immunogenic compositions are administered in a mannercompatible with the dosage formulation, and in such amount as to betherapeutically effective, protective and immunogenic. The quantity tobe administered depends on the subject to the immunized, including, forexample, the capacity of the subject's immune system to synthesizeantibodies, and if needed, to produce a cell-mediated, humoral orantibody-mediated immune response. Precise amounts of antigen andimmunogenic composition to be administered depend on the judgement ofthe practitioner. However, suitable dosage ranges are readilydeterminable by those skilled in the art and may be of the order ofmicrograms to milligrams. Suitable regimes for initial administrationand booster doses are also variable, but may include an initialadministration followed by subsequent administrations. The dosage of thevaccine may also depend on the route of administration and will varyaccording to the size of the host.

[0270] Passive immunity can be conferred to an animal subject suspectedof suffering an infection with streptococci by administering antiserum,polyclonal antibodies, or a neutralizing monoclonal antibody against oneor more Ema polypeptide of the invention to the patient. A combinationof antibodies directed against one or more Ema polypeptide selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE, in combination with one ormore of antibodies against Spb1, Spb2, Rib and C protein alpha antigenis also contemplated by the present invention. Although passive immunitydoes not confer long term protection, it can be a valuable tool for thetreatment of a bacterial infection in a subject who has not beenvaccinated. Passive immunity is particularly important fqr the treatmentof antibiotic resistant strains of bacteria, since no other therapy maybe available. Preferably, the antibodies administered for passive immunetherapy are autologous antibodies. For example, if the subject is ahuman, preferably the antibodies are of human origin or have been“humanized,” in order to minimize the possibility of an immune responseagainst the antibodies. The active or passive vaccines of the inventioncan be used to protect an animal subject from infection bystreptococcus, particularly Group B streptococcus.

[0271] Vaccines for GBS have been previously generated and tested.Preliminary vaccines used unconjuated purified polysaccaride. GBSpolysaccharides and oligosaccharides are poorly immunogenic and fail toelicit significant memory and booster responses. Baker et al immunized40 pregnant women with purified serotype III capsular polysaccharide(Baker, C. J. et al. (1998) New Engl J of Med 319:1180-1185). Overall,only 57% of women with low levels of specific antibody responded to thethe vaccine. The poor immunogenicity of purified polysaccharide antigenwas further demonstrated in a study in which thirty adult volunteerswere immunized with a tetravalent vaccine composed of purifiedpolysaccharide from serotypes Ia, Ib, II, and III (Kotloff, K. L. et al.(1996) Vaccine 14:446-450). Although safe, this vaccine was onlymodestly immunogenic, with only 13% of subjects responding to type Ib,17% to type II, 33% responding to type Ia, and 70% responding to typeIII polysaccharide. The poor immunogenicity of polysaccaride antigensprompted efforts to develop polysaccharide conjugate vaccines, wherebythese polysaccharides or oligosaccharides are conjugated to proteincarriers. Ninety percent of healthy adult women immunized with a typeIII polysaccharide-tetanus toxoid conjugate vaccine responded with a4-fold rise in antibody concentration, compared to 50% immunized withplain polysaccharide (Kasper, D. L. et al (1996) J of Clin Invest98:2308-2314). A type Ia/Ib polysaccharide-tetanus toxoid conjugatevaccine was similarly more immunogenic in healthy adults than plainpolysaccharide (Baker, C. J. et al (1999) J Infect Dis 179:142-150).

[0272] The general method for the conjugation of polysaccharide isdescribed in Wessels et al (Wessels, M. R. et al (1990) J ClinInvestigation 86: 1428-1433). Prior to coupling with tetanus toxoid,aldehyde groups are introduced on the polysaccharide by controlledperiodate oxidation, resulting in the conversion of a portion of thesialic acid residues of the polysaccharide to residues of the 8-carbonanalogue of sialic acid, 5-acetamido-3,5-dideoxy-D-galactosyloctulosonicacid. Tetanus toxoid is conjugated to the polysaccharide by reductiveamination using free aidehyde groups present on the partially oxidizedsialic acid residues. The preparation and conjugation ofoligosaccharides is described in Paoletti et al (Paoletti, L. C. et al(1990) J. Biol Chem 265: 18278-18283). Purified capsular polysaccharideis depolymerized by enzymatic digestion using endo-beta-galactosidaseproduced by Citrobacter freundii. Following digestion, oligosaccharidesare fractionated by gel filtration chromatography. Tetanus toxoid wascovalently coupled via a synthetic spacer molecule to the reducing endof the oligosaccharide by reductive amination.

[0273] Methods and vaccines comprising GBS conjugate vaccines,comprising capsular polysaccharide and protein are provided anddescribed in U.S. Pat. Nos. 5,993,825, 5,843,461, 5,795,580, 5,302,386and 4,356,263, which are incorporated herein by reference in theirentirety. These conjugate vaccines include polysaccharide-tetanus toxoidconjugate vaccines.

[0274] One polypeptide proposed to be utilized in a GBS vaccine is therepetitive GBS C protein alpha antigen, which contains up to ninetandemly repeated units of 82 amino acids (Michel, J. K. et al (1992)PNAS USA 89: 10060-10064). The polypeptide, methods and vaccinesthereof, including polysaccharide-conjugate vaccines generatedtherewith, are provided and described in U.S. Pat. Nos. 5,968,521,5,908,629, 5,858,362, 5,847,081, 5,843,461, 5,843,444, 5,820,860, and5,648,241, which are herein incorporated by reference in their entirety.Antibodies generated against C protein alpha antigen with a largenumbers of repeats protect against infection, but GBS are able to changethe structure of the protein by deleting one or more of the repeatregions and escape detection by these antibodies (Madoff, L. C. et al(1996) PNAS USA 93: 4131-4136). This effect could theoretically beprevented by immunization with a protein with a lower number of repeatunits, but the immunogenicity of the C protein alpha antigen isinversely related to the number of repeats 65% of mice responded toimmunization with the 9-repeat protein, but only 11% to a 1-repeatprotein (Gravekamp, C. et al (1997) Infect Immunity 65: 5216-5221). Thisis a disadvantage with any protein with a repetitive structure—it iscommon for bacteria to be able to alter or reassort these genes to alterthe proteins exposed on their surface.

[0275] Typical doses for a vaccine composed of a protein antigen are inthe range of 2.5-50 ug of total protein per dose. Typical doses for apolysaccharide-protein conjugate vaccine are 7.5-25 ug of polysaccharideand 1.25-250 ug of carrier protein. These types of vaccines are almostalways given intramuscularly. Dosing schedules of a vaccine can bereadily determined by the skilled artisan, particularly by comparison ofsimilar vaccines, including other GBS vaccines. If used as a universalvaccine, a GBS vaccine would be integrated into the routine immunizationschedule. Most similar vaccines require a primary series ofimmunizations (usually 2 or 3 doses at 2 month intervals beginning at 1or 2 months of age) and a single booster at 12-18 months of age. Asmaller number of doses or a single dose may be adequate in olderchildren (over a year of age). For immunization of pregnant women, anexemplary immunization schedule would be a single dose given in thesecond or early third trimester. For immunization of non-pregnantadults, a single dose would probably be used. The requirement forsubsequent booster doses in adults is difficult to predict this would bebased on the immunogenicity of the vaccine and ongoing surveillance ofvaccine efficacy.

Immunogenic Compositions

[0276] In a further aspect, the present invention provides animmunogenic composition comprising one of more bacterial Emapolypeptides. In a still further aspect, the present invention providesan immunogenic composition comprising one of more streptococcal Emapolypeptides. In a particular aspect, the present invention provides animmunogenic composition comprising one of more Group B streptococcalpolypeptides selected from the group of EmaA, EmaB, EmaC, EmaD, EmaE anda fragment thereof, and a pharmaceutically accpetable adjuvant.Immunogenic compositions may comprise a combination of one or more GroupB Ema polypeptide, or an immunogenic polypeptide fragment thereof, withone or more additional GBS polypeptide or GBS capsular polysaccharide oroligosaccharide.

[0277] The present invention further provides an immunogenic compositioncomprising one or more Group B streptococcal polypeptide selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE, further comprising one ormore antigens selected from the group of the polypeptide Spb1 or animmunogenic fragment thereof, the polypeptide Spb2 or an immunogenicfragment thereof, C protein alpha antigen or an immunogenic fragmentthereof, Rib or an immunogenic fragment thereof, and Group Bstreptococcal polysaccharides or oligosaccharides.

Pharmaceutical Compositions

[0278] The invention provides pharmaceutical compositions comprising abacterial Ema polypeptide, particularly a streptococcal Ema polypeptide,and a pharmaceutically acceptable carrier. The invention providespharmaceutical compositions comprising a Group B streptococcalpolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE,and a pharmaceutically acceptable carrier. The present invention furtherprovides pharmaceutical compositions comprising one or more GBS Emapolypeptide, or a fragment thereof, in combination with one or more ofGBS polypeptide Spb1, Spb2, C protein alpha antigen, Rib, a Group Bstreptococcal polysaccharide or oligosaccharide vaccine, and ananti-streptococcal vaccine.

[0279] Such pharmaceutical composition for preventing streptococcalattachment to mucosal surface may include antibody to Ema polypeptideEmaA, EmaB, EmaC, EmaD or EmaE or any combination of antibodies to oneor more such Ema polypeptide. In addition, any such composition mayfurther include antibody to GBS polypeptides Spb1, Spb2, C protein alphaantigen, or Rib. Blocking adherence using such antibody blocks theinitial step in infection thereby reducing colonization. This in turndecreases person to person transmission and prevents development ofsymptomatic disease.

[0280] The present invention provides a pharmaceutical compositioncomprising an antibody to a Group B streptococcal protein selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE, and a pharmaceuticallyacceptable carrier. The invention further provides a pharmaceuticalcomposition comprising a combination of at least two antibodies to GroupB streptococcal proteins and a pharmaceutically acceptable carrier,wherein at least one antibody to a protein selected from the group ofEmaA, EmaB, EmaC, EmaD, EmaE, is combined with at least one antibody toa protein selected from the group of Spb1, Spb2, Rib, and C proteinalpha antigen.

[0281] It is still a further object of the present invention to providea method for the prevention or treatment of mammals to control theamount or activity of streptococci, so as to treat or prevent theadverse consequences of invasive, spontaneous, or idiopathicpathological states.

[0282] It is still a further object of the present invention to providea method for the prevention or treatment of mammals to control theamount or activity of Group B streptococci, so as to treat or preventthe adverse consequences of invasive, spontaneous, or idiopathicpathological states.

[0283] The invention provides a method for preventing infection with abacterium that expresses a streptococcal Ema polypeptide comprisingadministering an immunogenically effective dose of a vaccine comprisingan Ema polypeptide selected from the group of EmaA, EmaB, EmaC, EmaD andEmaE to a subject.

[0284] The invention further provides a method for preventing infectionwith a bacterium that expresses a Group B streptococcal Ema polypeptidecomprising administering an immunogenically effective dose of a vaccinecomprising an Ema polypeptide selected from the group of EmaA, EmaB,EmaC, EmaD and EmaE to a subject.

[0285] The present invention is directed to a method for treatinginfection with a bacterium that expresses a Group B streptococcal Emapolypeptide comprising administering a therapeutically effective dose ofa pharmaceutical composition comprising an Ema polypeptide selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE, and a pharmaceuticallyacceptable carrier to a subject.

[0286] The invention further provides a method for treating infectionwith a bacterium that expresses a Group B streptococcal Ema polypeptidecomprising administering a therapeutically effective dose of apharmaceutical composition comprising an antibody to an Ema polypeptideselected from the group of EmaA, EmaB, EmaC, EmaD and EmaE, and apharmaceutically acceptable carrier to a subject.

[0287] In a further aspect, the invention provides a method of inducingan immune response in a subject which has been exposed to or infectedwith a Group B streptococcal bacterium comprising administering to thesubject an amount of the pharmaceutical composition comprising an Emapolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE,and a pharmaceutically acceptable carrier, thereby inducing an immuneresponse.

[0288] The invention still further provides a method for preventinginfection by a streptococcal bacterium in a subject comprisingadministering to the subject an amount of a pharmaceutical compositioncomprising an antibody to an Ema polypeptide selected from the group ofEmaA, EmaB, EmaC, EmaD and EmaE and a pharmaceutically acceptablecarrier or diluent, thereby preventing infection by a streptococcalbacterium.

[0289] The invention further provides an ema mutant bacteria which isnon-adherent and/or non-invasive to cells and which is mutated in one ormore genes selected from the group of emaA, emaB, emaC, emaD and emaE.Particularly, such ema mutant is a Group B streptococcal bacteria. Suchnon-adherent and/or non-invasive ema mutant bacteria can further beutilized in expressing other immunogenic or therapeutic proteins for thepurposes of eliciting immune responses to any such other proteins in thecontext of vaccines and in other forms of therapy.

[0290] This invention provides a method of inhibiting colonization ofhost cells in a subject which has been exposed to or infected with astreptococcal bacterium comprising administering to the subject anamount of a pharmaceutical composition comprising an Ema polypeptideselected from the group of EmaA, EmaB, EmaC, EmaD and EmaE, therebyinducing an immune response. The therapeutic peptide that blockscolonization is delivered by the respiratory mucosal. The pharmaceuticalcomposition comprises the polypeptide selected from the group of SEQ IDNO: 2, 4, 6, 8 and 10.

[0291] As used herein, “pharmaceutical composition” could meantherapeutically effective amounts of polypeptide products or antibodiesof the invention together with suitable diluents, preservatives,solubilizers, emulsifiers, adjuvant and/or carriers useful in therapyagainst bacterial infection or in inducing an immune response. A“therapeutically effective amount” as used herein refers to that amountwhich provides a therapeutic effect for a given condition andadministration regimen. Such compositions are liquids or lyophilized orotherwise dried formulations and include diluents of various buffercontent (e.g., Tris-HCl., acetate, phosphate), pH and ionic strength,additives such as albumin or gelatin to prevent absorption to surfaces,detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts),solubilizing agents (e.g., glycerol, polyethylene glycerol),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives(e.g., Thimerosal, benzyl alcohol, parabens), bulking substances ortonicity modifiers (e.g., lactose, mannitol), covalent attachment ofpolymers such as polyethylene glycol to the protein, complexation withmetal ions, or incorporation of the material into or onto particulatepreparations of polymeric compounds such as polylactic acid, polglycolicacid, hydrogels, etc, or onto liposomes, microemulsions, micelles,unilamellar or multilamellar vesicles, erythrocyte ghosts, orspheroplasts. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance of the polypeptides of the present invention. The choice ofcompositions will depend on the physical and chemical properties of thepolypeptide. Controlled or sustained release compositions includeformulation in lipophilic depots (e.g., fatty acids, waxes, oils). Alsocomprehended by the invention are particulate compositions coated withpolymers (e.g., poloxamers or poloxamines) and the polypeptides of thepresent invention coupled to antibodies directed against tissue-specificreceptors, ligands or antigens or coupled to ligands of tissue-specificreceptors. Other embodiments of the compositions of the inventionincorporate particulate forms, protective coatings, protease inhibitorsor permeation enhancers for various routes of administration, includingparenteral, pulmonary, nasal and oral.

[0292] Further, as used herein “pharmaceutically acceptable carrier” arewell known to those skilled in the art and include, but are not limitedto, 0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline.Additionally, such pharmaceutically acceptable carriers may be aqueousor non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, collating agents, inertgases and the like.

[0293] The phrase “pharmaceutically acceptable” refers to molecularentities and compositions that are physiologically tolerable and do nottypically produce an allergic or similar untoward reaction, such asgastric upset, dizziness and the like, when administered to a human.

[0294] The phrase “therapeutically effective amount” is used herein tomean an amount sufficient to prevent, and preferably reduce by at leastabout 30 percent, more preferably by at least 50 percent, mostpreferably by at least 90 percent, a clinically significant infection bystreptococcal bacterium. Alternatively, in the case of a vaccine orimmunogenic composition, a therapeutically effective amount is usedherein to mean an amount sufficient and suitable to elicit an immuneresponse and antibody response in an individual, and particularly toprovide a response sufficient to prevent, and preferably reduce by atleast about 30 percent, more preferably by at least 50 percent, mostpreferably by at least 90 percent, a clinically significant infection bystreptococcal bacterium.

[0295] Controlled or sustained release compositions include formulationin lipophilic depots (e.g. fatty acids, waxes, oils). Also comprehendedby the invention are particulate compositions coated with polymers (e.g.poloxamers or poloxamines) and the compound coupled to antibodiesdirected against tissue-specific receptors, ligands or antigens orcoupled to ligands of tissue-specific receptors. Other embodiments ofthe compositions of the invention incorporate particulate formsprotective coatings, protease inhibitors or permeation enhancers forvarious routes of administration, including parenteral, pulmonary, nasaland oral.

[0296] When administered, compounds are often cleared rapidly frommucosal surfaces or the circulation and may therefore elicit relativelyshort-lived pharmacological activity. Consequently, frequentadministrations of relatively large doses of bioactive compounds may byrequired to sustain therapeutic efficacy. Compounds modified by thecovalent attachment of water-soluble polymers such as polyethyleneglycol, copolymers of polyethylene glycol and polypropylene glycol,carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinylpyrrolidone or polyproline are known to exhibit substantiallylonger half-lives in blood following intravenous injection than do thecorresponding umnodified compounds (Abuchowski et al., 1981; Newmark etal., 1982; and Katre et al., 1987). Such modifications may also increasethe compound's solubility in aqueous solution, eliminate aggregation,enhance the physical and chemical stability of the compound, and greatlyreduce the immunogenicity and reactivity of the compound. As a result,the desired in vivo biological activity may be achieved by theadministration of such polymer-compound abducts less frequently or inlower doses than with the unmodified compound.

[0297] Dosages. The sufficient amount may include but is not limited tofrom about 1 μg/kg to about 1000 mg/kg. The amount may be 10 mg/kg. Thepharmaceutically acceptable form of the composition includes apharmaceutically acceptable carrier.

[0298] As noted above, the present invention provides therapeuticcompositions comprising pharmaceutical compositions comprising vectors,vaccines, polypeptides, nucleic acids and antibodies, anti-antibodies,and agents, to compete with the Group B streptococcus bacterium forpathogenic activities, such as adherence to host cells.

[0299] The preparation of therapeutic compositions which contain anactive component is well understood in the art. Typically, suchcompositions are prepared as an aerosol of the polypeptide delivered tothe nasopharynx or as injectables, either as liquid solutions orsuspensions, however, solid forms suitable for solution in, orsuspension in, liquid prior to injection can also be prepared. Thepreparation can also be emulsified. The active therapeutic ingredient isoften mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents which enhance the effectivenessof the active ingredient.

[0300] An active component can be formulated into the therapeuticcomposition as neutralized pharmaceutically acceptable salt forms.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide or antibodymolecule) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed from thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0301] A composition comprising “A” (where “A” is a single protein, DNAmolecule, vector, etc.) is substantially free of “B” (where “B”comprises one or more contaminating proteins, DNA molecules, vectors,etc.) when at least about 75% by weight of the proteins, DNA, vectors(depending on the category of species to which A and B belong) in thecomposition is “A”. Preferably, “A” comprises at least about 90% byweight of the A+B species in the composition, most preferably at leastabout 99% by weight.

[0302] The phrase “therapeutically effective amount” is used herein tomean an amount sufficient to reduce by at least about 15 percent,preferably by at least 50 percent, more preferably by at least 90percent, and most preferably prevent, a clinically significant deficitin the activity, function and response of the host. Alternatively, atherapeutically effective amount is sufficient to cause an improvementin a clinically significant condition in the host. In the context of thepresent invention, a deficit in the response of the host is evidenced bycontinuing or spreading bacterial infection. An improvement in aclinically significant condition in the host includes a decrease inbacterial load, clearance of bacteria from colonized host cells,reduction in fever or inflammation associated with infection, or areduction in any symptom associated with the bacterial infection.

[0303] According to the invention, the component or components of atherapeutic composition of the invention may be introduced parenterally,transmucosally, e.g., orally, nasally, pulmonarailly, or rectally, ortransdermally. Preferably, administration is parenteral, e.g., viaintravenous injection, and also including, but is not limited to,intra-arteriole, intramuscular, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial administration. Oralor pulmonary delivery may be preferred to activate mucosal immunity;since Group B streptococci generally colonize the nasopharyngeal andpulmonary mucosa, particularly that of neonates, mucosal immunity may bea particularly effective preventive treatment. The term “unit dose” whenused in reference to a therapeutic composition of the present inventionrefers to physically discrete units suitable as unitary dosage forhumans, each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent; i.e., carrier, or vehicle.

[0304] In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317327; see generally ibid).

[0305] In yet another embodiment, the therapeutic compound can bedelivered in a controlled release system. For example, the polypeptidemay be administered using intravenous infusion, an implantable osmoticpump, a transdermal patch, liposomes, or other modes of administration.In one embodiment, a pump may be used (see Langer, supra; Sefton, CRCCrit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507.(1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In anotherembodiment, polymeric materials can be used (see Medical Applications ofControlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.(1974); Controlled Drug Bioavailability, Drug Product Design andPerformance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger andPeppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see alsoLevy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351(1989); Howard et al, J. Neurosurg. 71:105 (1989)). In yet anotherembodiment, a controlled release system can be placed in proximity ofthe therapeutic target, i.e., the brain, thus requiring only a fractionof the systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984)). Preferably, acontrolled release device is introduced into a subject in proximity ofthe site of inappropriate immune activation or a tumor. Other controlledrelease systems are discussed in the review by Langer (Science249:1527-1533 (1990)).

[0306] A subject in whom administration of an active component as setforth above is an effective therapeutic regimen for a bacterialinfection is preferably a human, but can be any animal. Thus, as can bereadily appreciated by one of ordinary skill in the art, the methods andpharmaceutical compositions of the present invention are particularlysuited to administration to any animal, particularly a mammal, andincluding, but by no means limited to, domestic animals, such as felineor canine subjects, farm animals, such as but not limited to bovine,equine, caprine, ovine, and porcine subjects, wild animals (whether inthe wild or in a zoological garden), research animals, such as mice,rats, rabbits, goats, sheep, pigs, dogs, cats, etc., i.e., forveterinary medical use.

[0307] In the therapeutic methods and compositions of the invention, atherapeutically effective dosage of the active component is provided. Atherapeutically effective dosage can be determined by the ordinaryskilled medical worker based on patient characteristics (age, weight,sex, condition, complications, other diseases, etc.), as is well knownin the art. Furthermore, as further routine studies are conducted, morespecific information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age and generalhealth of the recipient, is able to ascertain proper dosing. Generally,for intravenous injection or infusion, dosage may be lower than forintraperitoneal, intramuscular, or other route of administration. Thedosing schedule may vary, depending on the circulation half-life, andthe formulation used. The compositions are administered in a mannercompatible with the dosage formulation in the therapeutically effectiveamount. Precise amounts of active ingredient required to be administereddepend on the judgment of the practitioner and are peculiar to eachindividual. However, suitable dosages may range from about 0.1 to 20,preferably about 0.5 to about 10, and more preferably one to several,milligrams of active ingredient per kilogram body weight of individualper day and depend on the route of administration. Suitable regimes forinitial administration and booster shots are also variable, but aretypified by an initial administration followed by repeated doses at oneor more hour intervals by a subsequent injection or otheradministration.

[0308] Alternatively, continuous intravenous infusion sufficient tomaintain concentrations of ten nanomolar to ten micromolar in the bloodare contemplated. Administration with other compounds. For treatment ofa bacterial infection, one may administer the present active componentin conjunction with one or more pharmaceutical compositions used fortreating bacterial infection, including but not limited to (1)antibiotics; (2) soluble carbohydrate inhibitors of bacterial adhesin;(3) other small molecule inhibitors of bacterial adhesin; (4) inhibitorsof bacterial metabolism, transport, or transformation; (5) stimulatorsof bacterial lysis, or (6) anti-bacterial antibodies or vaccinesdirected at other bacterial antigens. Other potential active componentsinclude anti-inflammatory agents, such as steroids and non-steroidalanti-inflammatory drugs. Administration may be simultaneous (forexample, administration of a mixture of the present active component andan antibiotic), or may be in seriatim.

[0309] Accordingly, in specific embodiment, the therapeutic compositionsmay further include an effective amount of the active component, and oneor more of the following active ingredients: an antibiotic, a steroid,etc.

[0310] Thus, in a specific instance where it is desired to reduce orinhibit the infection resulting from a bacterium mediated binding ofbacteria to a host cell, or an antibody thereto, or a ligand thereof oran antibody to that ligand, the polypeptide is introduced to block theinteraction of the bacteria with the host cell.

[0311] Also contemplated herein is pulmonary delivery of an inhibitor ofthe polypeptide of the present invention having which acts as adhesininhibitory agent (or derivatives thereof). The adhesin inhibitory agent(or derivative) is delivered to the lungs of a mammal, where it caninterfere with bacterial, i.e., streptococcal, and preferably Group Bstreptococcal binding to host cells. Other reports of preparation ofproteins for pulmonary delivery are found in the art [Adjei et al.(1990)Pharmaceutical Research, 7:565-569; Adjei et al. (1990) InternationalJournal of Pharmaceutics, 63:135-144 (leuprolide acetate); Braquet et al(1989), Journal of Cardiovascular Pharmacology, 13(suppl. 5):143-146(endothelin-1); Hubbard et al(1989) Animals of Internal Medicine, Vol.III, pp. 206-212 (α1-antitrypsin); Smith et al. (1989) J. Clin. Invest.84:1145-1146 (α-1-proteinase); Oswein et al., “Aerosolization ofProteins”, Proceedings of Symposium on Respiratory Drug Delivery II,Keystone, Colorado, March, (1990) (recombinant human growth hormone);Debs et al. (1988) J. Immunol. 140:3482-3488 (interferon-γ and tumornecrosis factor alpha); Platz et al., U.S. Pat. No. 5,284,656(granulocyte colony stimulating factor)]. A method and composition forpulmonary delivery of drugs is described in U.S. Pat. No. 5,451,569,issued Sep. 19, 1995 to Wong et al.

[0312] All such devices require the use of formulations suitable for thedispensing of adhesin inhibitory agent (or derivative). Typically, eachformulation is specific to the type of device employed and may involvethe use of an appropriate propellant material, in addition to the usualdiluents, adjuvant and/or carriers useful in therapy. Also, the use ofliposomes, microcapsules or microspheres, inclusion complexes, or othertypes of carriers is contemplated. Chemically modified adhesininhibitory agent may also be prepared in different formulationsdepending on the type of chemical modification or the type of deviceemployed.

[0313] Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise adhesin inhibitory agent (orderivative) dissolved in water at a concentration of about 0.1 to 25 mgof biologically active adhesin inhibitory agent per ml of solution. Theformulation may also include a buffer and a simple sugar (e.g., foradhesin inhibitory agent stabilization and regulation of osmoticpressure). The nebulizer formulation may also contain a surfactant, toreduce or prevent surface induced aggregation of the adhesin inhibitoryagent caused by atomization of the solution in forming the aerosol.

[0314] Formulations for use with a metered-dose inhaler device willgenerally comprise a finely divided powder containing the adhesininhibitory agent (or derivative) suspended in a propellant with the aidof a surfactant. The propellant may be any conventional materialemployed for this purpose, such as a chlorofluorocarbon, ahydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

[0315] The liquid aerosol formulations contain adhesin inhibitory agentand a dispersing agent in a physiologically acceptable diluent. The drypowder aerosol formulations of the present invention consist of a finelydivided solid form of adhesin inhibitory agent and a dispersing agent.With either the liquid or dry powder aerosol formulation, theformulation must be aerosolized. That is, it must be broken down intoliquid or solid particles in order to ensure that the aerosolized doseactually reaches the mucous membranes of the nasal passages or the lung.The term “aerosol particle” is used herein to describe the liquid orsolid particle suitable for nasal or pulmonary administration, i.e.,that will reach the mucous membranes. Other considerations, such asconstruction of the delivery device, additional components in theformulation, and particle characteristics are important. These aspectsof pulmonary administration of a drug are well known in the art, andmanipulation of formulations, aerosolization means and construction of adelivery device require at most routine experimentation by one ofordinary skill in the art. In a particular embodiment, the mass mediandynamic diameter will be 5 micrometers or less in order to ensure thatthe drug particles reach the lung alveoli [Wearley, L. L. (1991) Crit.Rev. in Ther. Drug Carrier Systems 8:333].

[0316] Systems of aerosol delivery, such as the pressurized metered doseinhaler and the dry powder inhaler are disclosed in Newman, S. P.,Aerosols and the Lung, Clarke, S. W. and Davia, D. editors, pp. 197-22and can be used in connection with the present invention.

[0317] In a further embodiment, as discussed in detail infra, an aerosolformulation of the present invention can include other therapeuticallyor pharmacologically active ingredients in addition to adhesininhibitory agent, such as but not limited to an antibiotic, a steroid, anon-steroidal anti-inflammatory drug, etc.

[0318] Liquid Aerosol Formulations. The present invention providesaerosol formulations and dosage forms for use in treating subjectssuffering from bacterial, e.g., streptococcal, in particularlystreptococcal, infection. In general such dosage forms contain adhesininhibitory agent in a pharmaceutically acceptable diluent.Pharmaceutically acceptable diluents include but are not limited tosterile water, saline, buffered saline, dextrose solution, and the like.In a specific embodiment, a diluent that may be used in the presentinvention or the pharmaceutical formulation of the present invention isphosphate buffered saline, or a buffered saline solution generallybetween the pH 7.0-8.0 range, or water.

[0319] The liquid aerosol formulation of the present invention mayinclude, as optional ingredients, pharmaceutically acceptable carriers,diluents, solubilizing or emulsifying agents, surfactants andexcipients. The formulation may include a carrier. The carrier is amacromolecule which is soluble in the circulatory system and which isphysiologically acceptable where physiological acceptance means thatthose of skill in the art would accept injection of said carrier into apatient as part of a therapeutic regime. The carrier preferably isrelatively stable in the circulatory system with an acceptable plasmahalf life for clearance. Such macromolecules include but are not limitedto Soya lecithin, oleic acid and sorbitan trioleate, with sorbitantrioleate preferred.

[0320] The formulations of the present embodiment may also include otheragents useful for pH maintenance, solution stabilization, or for theregulation of osmotic pressure. Examples of the agents include but arenot limited to salts, such as sodium chloride, or potassium chloride,and carbohydrates, such as glucose, galactose or mannose, and the like.

[0321] The present invention further contemplates liquid aerosolformulations comprising adhesin inhibitory agent and anothertherapeutically effective drug, such as an antibiotic, a steroid, anon-steroidal anti-inflammatory drug, etc.

[0322] Aerosol Dry Powder Formulations. It is also contemplated that thepresent aerosol formulation can be prepared as a dry powder formulationcomprising a finely divided powder form of adhesin inhibitory agent anda dispersant.

[0323] Formulations for dispensing from a powder inhaler device willcomprise a finely divided dry powder containing adhesin inhibitory agent(or derivative) and may also include a bulking agent, such as lactose,sorbitol, sucrose, or mannitol in amounts which facilitate dispersal ofthe powder from the device, e.g., 50 to 90% by weight of theformulation. The adhesin inhibitory agent (or derivative) should mostadvantageously be prepared in particulate form with an average particlesize of less than 10 mm (or microns), most preferably 0.5 to 5 mm, formost effective delivery to the distal lung. In another embodiment, thedry powder formulation can comprise a finely divided dry powdercontaining adhesin inhibitory agent, a dispersing agent and also abulking agent. Bulking agents useful in conjunction with the presentformulation include such agents as lactose, sorbitol, sucrose, ormannitol, in amounts that facilitate the dispersal of the powder fromthe device.

[0324] The present invention further contemplates dry powderformulations comprising adhesin inhibitory agent and anothertherapeutically effective drug, such as an antibiotic, a steroid, anon-steroidal anti-inflammatory drug, etc.

[0325] Contemplated for use herein are oral solid dosage forms, whichare described generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which isherein incorporated by reference. Solid dosage forms include tablets,capsules, pills, troches or lozenges, cachets or pellets. Also,liposomal or proteinoid encapsulation may be used to formulate thepresent compositions (as, for example, proteinoid microspheres reportedin U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and theliposomes may be derivatized with various polymers (e.g., U.S. Pat. No.5,013,556). A description of possible solid dosage forms for thetherapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited byG. S. Banker and C. T. Rhodes Chapter 10, 1979, herein incorporated byreference. In general, the formulation will include the component orcomponents (or chemically modified forms thereof) and inert ingredientswhich allow for protection against the stomach environment, and releaseof the biologically active material in the intestine.

[0326] Also specifically contemplated are oral dosage forms of the abovederivatized component or components. The component or components may bechemically modified so that oral delivery of the derivative isefficacious. Generally, the chemical modification contemplated is theattachment of at least one moiety to the component molecule itself,where said moiety permits (a) inhibition of proteolysis; and (b) uptakeinto the blood stream from the stomach or intestine. Also desired is theincrease in overall stability of the component or components andincrease in circulation time in the body. Examples of such moietiesinclude: polyethylene glycol, copolymers of ethylene glycol andpropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, 1981,“Soluble Polymer-Enzyme Abducts” In: Enzymes as Drugs, Hocenberg andRoberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383; Newmark,et al. (1982) J. Appl. Biochem. 4:185-189. Other polymers that could beused are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred forpharmaceutical usage, as indicated above, are polyethylene glycolmoieties.

[0327] For the component (or derivative) the location of release may bethe stomach, the small intestine (the duodenum, the jejunem, or theileum), or the large intestine. One skilled in the art has availableformulations which will not dissolve in the stomach, yet will releasethe material in the duodenum or elsewhere in the intestine. Preferably,the release will avoid the deleterious effects of the stomachenvironment, either by protection of the protein (or derivative) or byrelease of the biologically active material beyond the stomachenvironment, such as in the intestine.

[0328] To ensure full gastric resistance a coating impermeable to atleast pH 5.0 is essential. Examples of the more common inert ingredientsthat are used as enteric coatings are cellulose acetate trimellitate(CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric,cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac.These coatings may be used as mixed films.

[0329] A coating or mixture of coatings can also be used on tablets,which are not intended for protection against the stomach. This caninclude sugar coatings, or coatings which make the tablet easier toswallow. Capsules may consist of a hard shell (such as gelatin) fordelivery of dry therapeutic i.e. powder; for liquid forms, a softgelatin shell may be used. The shell material of cachets could be thickstarch or other edible paper. For pills, lozenges, molded tablets ortablet triturates, moist massing techniques can be used.

[0330] The peptide therapeutic can be included in the formulation asfine multiparticulates in the form of granules or pellets of particlesize about 1 mm. The formulation of the material for capsuleadministration could also be as a powder, lightly compressed plugs oreven as tablets. The therapeutic could be prepared by compression.

[0331] Colorants and flavoring agents may all be included. For example,the protein (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

[0332] One may dilute or increase the volume of the therapeutic with aninert material. These diluents could include carbohydrates, especiallymannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modifieddextran and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

[0333] Disintegrants may be included in the formulation of thetherapeutic into a solid dosage form. Materials used as disintegratesinclude but are not limited to starch, including the commercialdisintegrant based on starch, Explotab. Sodium starch glycolate,Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodiumalginate, gelatin, orange peel, acid carboxymethyl cellulose, naturalsponge and bentonite may all be used. Another form of the disintegrantsare the insoluble cationic exchange resins. Powdered gums may be used asdisintegrants and as binders and these can include powdered gums such asagar, Karaya or tragacanth. Alginic acid and its sodium salt are alsouseful as disintegrants. Binders may be used to hold the therapeuticagent together to form a hard tablet and include materials from naturalproducts such as acacia, tragacanth, starch and gelatin Others includemethyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose(CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose(HPMC) could both be used in alcoholic solutions to granulate thetherapeutic.

[0334] An antifrictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

[0335] Glidants that might improve the flow properties of the drugduring formulation and to aid rearrangement during compression might beadded. The glidants may include starch, talc, pyrogenic silica andhydrated silicoaluminate.

[0336] To aid dissolution of the therapeutic into the aqueousenvironment a surfactant might be added as a wetting agent. Surfactantsmay include anionic detergents such as sodium lauryl sulfate, dioctylsodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergentsmight be used and could include benzalkonium chloride or benzethomiumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

[0337] Additives which potentially enhance uptake of the polypeptide (orderivative) are for instance the fatty acids oleic acid, linoleic acidand linolenic acid.

[0338] Pulmonary Delivery. Also contemplated herein is pulmonarydelivery of the present polypeptide (or derivatives thereof). Thepolypeptide (or derivative) is delivered to the lungs of a mammal whileinhaling and coats the mucosal surface of the alveoli. Other reports ofthis include Adjei et al. (1990) Pharmaceutical Research 7:565-569;Adjei et al. (1990) International Journal of Pharmaceutics 63:135-144(leuprolide acetate); Braquet et al. (1989) Journal of CardiovascularPharmacology, 13 (suppl. 5):143-146 (endothelin-1); Hubbard et al.(1989) Annals of Internal Medicine, Vol. III, pp. 206-212(a1-antitrypsin); Smith et al (1989) J. Clin. Invest. 84:1145-1146(a-1-proteinase); Oswein et al. (1990) “Aerosolization of Proteins”,Proceedings of Symposium on Respiratory Drug Delivery II, Keystone,Colorado, March, (recombinant human growth hormone); Debs et al. (1988)J. Immunol. 140:3482-3488 (interferon-g and tumor necrosis factor alpha)and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colonystimulating factor). A method and composition for pulmonary delivery ofdrugs for systemic effect is described in U.S. Pat. No. 5,451,569,issued Sep. 19, 1995 to Wong et al.

[0339] Contemplated for use in the practice of this invention are a widerange of mechanical devices designed for pulmonary delivery oftherapeutic products, including but not limited to nebulizers, metereddose inhalers, and powder inhalers, all of which are familiar to thoseskilled in the art.

[0340] Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise polypeptide (or derivative)dissolved in water at a concentration of about 0.1 to 25 mg ofbiologically active protein per mL of solution. The formulation may alsoinclude a buffer and a simple sugar (e.g., for protein stabilization andregulation of osmotic pressure). The nebulizer formulation may alsocontain a surfactant, to reduce or prevent surface induced aggregationof the protein caused by atomization of the solution in forming theaerosol.

[0341] Formulations for use with a metered-dose inhaler device willgenerally comprise a finely divided powder containing the polypeptide(or derivative) suspended in a propellant with the aid of a surfactant.The propellant may be any conventional material employed for thispurpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol, and1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactantsinclude sorbitan trioleate and soya lecithin. Oleic acid may also beuseful as a surfactant.

[0342] Formulations for dispensing from a powder inhaler device willcomprise a finely divided dry powder containing polypeptide (orderivative) and may also include a bulking agent, such as lactose,sorbitol, sucrose, or mannitol in amounts which facilitate dispersal ofthe powder from the device, e.g., 50 to 90% by weight of theformulation. The protein (or derivative) should most advantageously beprepared in particulate form with an average particle size of less than10 mm (or microns), most preferably 0.5 to 5 mm, for most effectivedelivery to the distal lung.

[0343] Nasal Delivery. Nasal or nasopharyngeal delivery of thepolypeptide (or derivative) is also contemplated. Nasal delivery allowsthe passage of the polypeptide directly over the upper respiratory tractmucosal after administering the therapeutic product to the nose, withoutthe necessity for deposition of the product in the lung. Formulationsfor nasal delivery include those with dextran or cyclodextran tidenomenclature, J. Biol. Chem., 243:3552-59 (1969), abbreviations foramino acid

[0344] The therapeutic polypeptide-, analog- or activefragment-containing compositions are conventionally administeredintravenously, as by injection of a unit dose, for example. The term“unit dose” when used in reference to a therapeutic composition of thepresent invention refers to physically discrete units suitable asunitary dosage for humans, each unit containing a predetermined quantityof active material calculated to produce the desired therapeutic effectin association with the required diluent; i.e., carrier, or vehicle.

[0345] The compositions are administered in a manner compatible with thedosage formulation, and in a therapeutically effective amount. Thequantity to be administered depends on the subject to be treated,capacity of the subject's immune system to utilize the activeingredient, and degree of inhibition or neutralization of˜bindingcapacity desired. Precise amounts of active ingredient required to beadministered depend on the judgment of the practitioner and are peculiarto each individual. However, suitable dosages may range from about 0.1to 20, preferably about 0.5 to about 10, and more preferably one toseveral, milligrams of active ingredient per kilogram body weight ofindividual per day and depend on the route of administration. Suitableregimes for initial administration and booster shots are also variable,but are typified by an initial administration followed by repeated dosesat one or more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations of ten nanomolar to ten micromolarin the blood are contemplated.

[0346] The invention may be better understood by reference to thefollowing non-limiting Examples, which are provided as exemplary of theinvention. The following examples are presented in order to more fullyillustrate the preferred embodiments of the invention and should in noway be construed, however, as limiting the broad scope of the invention.

EXAMPLE 1 Identification of Group B Streptococcus Genes

[0347] Comparing the genetic and phenotypic composition ofgenetically-related groups of a bacterial species facilitatesidentifying virulence factors present in the most pathogenic groups.Type III GBS can be subdiyided into three groups of related strainsbased on the analysis of restriction digest patterns (RDPs) produced bydigestion of chromosomal DNA with Hind III and Sse 8387 (5, 6). Over 90%of invasive type III GBS disease in neonates in Japan and in Salt LakeCity is caused by bacteria from one of three RDP types, termed RDP typeIII-3, while RDP type III-2 are significantly more likely to be isolatedfrom vagina than from blood or CSF (6). These results suggest that thisgenetically-related cluster of type III-3 GBS are more virulent thanIII-2 strains and could be responsible for the majority of invasive typeIII disease globally. We proposed that bacterial factors that contributeto the increased virulence of III-3 strains can be identified bycharacterizing the differences between the genetic composition of III-3and III-2 strains. Such genetic differences will be found in thebacterial chromosomes since these strains do not contain plasmids (6).

[0348] To identify genes present in virulent type III-3 GBS strains andnot in the avirulent type III-2 strains we used a modification of thetechnique described by Lisitsyn et al (7). High molecular weight genomicDNA from an invasive RDP type III-3 GBS strain (strain 874391) and acolonizing (“avirulent”) RDP type III-2 strain (strain 865043) wasprepared by cell lysis with mutanolysin and Proteinase K digestion (5).For genetic subtraction, genomic DNA from both strains was digestedwithTaq I. Taq I-digested DNA from the virulent strain was mixed withtwo complementary oligonucleotides (TaqA (5′-CTAGGTGGATCCTTCGGCAAT-3′(SEQ ID NO: 11)) and TaqB (5′-CGATTGCCGA-3′ (SEQ ID NO: 12)), heated to50° C. for 5 minutes, then allowed to cool slowly to 16° C. in T4 ligasebuffer. Oligonucleotides were ligated to the virulent strain DNA byincubation with 20 units of T4 ligase at 16° C. for 12 hours. Afterligation, 500 ng of DNA from the virulent strain, with ligated linkers,and 40 ug of DNA from the avirulent strain, without linkers, was mixedtogether, denatured by heating, and hybridized at 68° C. for 20 hours.

[0349] Ten percent of the resulting hybridization mixture was incubatedwith Taq DNA polymerase and dNTPs to fill in the ends of annealedvirulent strain DNA. The hybridized DNA was amplified by Taq DNApolymerase for 10 cycles using the TaqA oligonucleotide as the forwardand reverse amplification primer. After amplification, single strandedproducts remaining after amplification were digested with mung beannuclease. Twenty percent of the resulting product was then reamplifiedfor 20 cycles. This process of subtraction followed by PCR amplificationresults in enhanced amplification of DNA segments from the III-3 strainsthat do not hybridize with DNA segments from the III-2 strains.

[0350] A total of four cycles of subtraction and amplification werecarried out, using successively smaller quantities of III-3 specific PCRproducts and alternating two sets of adaptors (TaqA/B (SEQ ID NOS: 11and 12, respectively) and TaqE/F (TaqE (5′-AGGCAACTGTGCTAACCGAGGGAAT-3′(SEQ ID NO: 13)); and TaqF (5′-CGATTCCCTCG-3′ (SEQ ID NO: 14)). Thefinal amplification products were ligated into pBS KS+ vectors. Thirteenclones were randomly selected for analysis. These probes were used inslot and dot blot experiments to determine whether subtraction wassuccessful and to identify probes hybridizing with all III-3 strains.Each of the 6 unique probes hybridized with the parental III-3 virulentstrain, while none of the probes hybridized with the avirulent III-2strains. Two of the amplified sequence tags (clones DY1-1 and DY1-11)hybridized with genomic DNA from all 62 type III isolates, but did nothybridize with DNA prepared from the III-2 and III-1 isolates (FIG. 1).To obtain additional sequence information, we constructed a genomic GBSIII-3 library. Multiple plaques hybridizing with each of the III-3GBS-specific probes have been purified for further characterization.

[0351] Results

[0352] The spb Locus

[0353] Three overlapping genomic clones hybridizing with probe DY1-1were identified. A 6.4 kb Sal I-Bgl II fragment present in each clonewas subcloned and sequenced. This genomic DNA is present in all RDP typeIII-3 strains but not in serotype III-2, III-1 or other GBS serotypestrains.

[0354] Over 90% of this genomic DNA fragment has been sequenced andfound to contain 5 open reading frames (ORFs). Two ORFs appear to becandidates for virulence genes. spb1 is a 1509 bp ORF. The predictedprotein (502 amino acids and Mr 53,446) has the characteristics of acell-wall bound protein. The nucleic acid and predicted amino acidsequences of sbp1 are provided in SEQ ID NOS: 15 and 16, respectively.The N-terminus of the predicted protein is a hydrophilic, basic stretchof 6 amino acids followed by a 23 amino acid hydrophobic, proline-richcore, consistent with a signal peptide. The hydrophilic mature proteinterminates in a typical LPXTG (SEQ ID NO: 17) domain that immediatelyprecedes a hydrophobic 20 amino acid core and a short, basic hydrophilicterminus. The nucleotide sequence is not homologous to sequences ofother known bacterial genes. The translated amino acid sequence,however, shares segmental homology with a number of characterizedproteins, including the fimbrial type 2 protein of Actinomycesnaeslundii (27% identity over 350 amino acids) and the fimbrial type 1protein of Actinomyces viscosus (25% homology over 420 amino acids)(16), the T6 surface protein of S. pyogenes (23% identity over 359 aminoacids) (20), and the hsf (27% identity over 260 amino acids) and HMW1adhesins (25% identity over 285 amino acids) of Haemophilus influenzae(21, 22). The function of the S. pyogenes T6 protein is unknown. Each ofthe other homologs plays a role in bacterial adhesion and/or invasion.

[0355] A spb1⁻ isogenic deletion mutant GBS strain was created byhomologous recombination (using the method as described in Example 2below) and the ability of the spb1⁻ mutant to adhere to and invade A549respiratory epithelial cells was determined. Compared to the wild typestrain, the number of spb1⁻ bacteria adherent to A549 monolayers wasreduced by 60.0% (p<0.01) and the number of intracellular invadingbacteria was reduced by 53.6% (p<0.01). This data suggests spb1 maycontribute to the pathogenesis of GBS pneumonia and bacterial entry intothe bloodstream.

[0356] The second ORF, spb2, terminates 37 bp upstream from spb1 and isin the same transcriptional orientation. This 1692 bp ORF has a deducedamino acid sequence of 579 residues and Mr 64,492. The nucleic acid andpredicted amino acid sequences of sbp2 are provided in SEQ ID NOS: 18and 19, respectively. spb2 shares 50.5% nucleic acid identity and 20.7%amino acid identity with spb1. Conservation is highest in thecarboxy-terminal regions, including a shared LPSTGG (SEQ ID NO: 20)motif. In contrast to spb1, spb2 does not have a obvious signalsequence. Its secretion may be mediated by carboxy-terminal recognitionsequences or by accessory peptides (23). The deduced amino acid sequenceof Spb2 is also homologous with S. pyogenes T6 and Actinomycesnaeslundii proteins, and to Listeria monocytogenes internalin A (22%identity over 308 amino acids); again, proteins important in adhesionand invasion (24).

[0357] The ema Locus

[0358] Two genomic clones hybridizing with probe DY1-11 were identified.A 7 kb Hind III fragment present in each clone was subcloned andsequenced. Unlike the serotype III specific spb sequences, this genomicDNA, which is adjacent to a region of serotype III-3 specific DNA, wasfound to be present in all GBS tested to date, including serotype Ia,Ib, II and V strains. This region of the GBS chromosome, which we havedesignated the extracellular matrix adhesin (ema) locus, contains 5significant ORFs.

[0359] emaA

[0360] The first ORF, emaA, is 738 bp long, with a predicted proteinproduct of 246 amino acids and Mr 26.2. The nucleic acid sequence (SEQID NO: 1) and predicted amino acid sequence (SEQ ID NO: 2) of emaA areshown in FIG. 2. The EmaA protein is a non-repetitive protein. The 27amino acid N-terminus of the predicted protein is consistent with asignal peptide. The mature protein has an imperfect cell wall bindingdomain (XPXTGG (SEQ ID NO:21)) followed by a transmembrane spanningdomain encompassing residues 219-235 and a terminal hydrophilic tail.The emaA nucleotide sequence is not homologous to known sequences ofbacterial genes. The translated amino acid sequence, however, sharessegmental homology with a number of characterized proteins, including acollagen adhesin, Bbp, of Staphylococcus aureus (37% identity over 103aa) (15), a type 2 fimbrial structural subunit of Actinomyces naeslandii(39% homology over 112 aa) (16), and the FimP protein of Actinomycesviscostis (28% homology over 228 aa) (17). The function of the S.pyogenes T6 protein is unknown. The type 1 and type 2 fimbria ofActinomyces mediate bacterial adhesion to salivary glycoproteins andvarious host cells, contributing to the pathogenesis of dental caries.

[0361] emaB

[0362] The second ORF, emaB, begins 94 bp 3′ of emaA and is in the sametranscriptional orientation. The nucleic acid sequence (SEQ ID NO: 3)and predicted amino acid sequence (SEQ ID NO: 4) of emaB are shown inFIG. 3. It is 924 bp long, with a predicted protein product of 308 aminoacids and Mr 33.9. The predicted EmaB protein is a nonrepetitiveprotein. The 27 amino acids N-terminus of the predicted protein isconsistent with a signal peptide. The mature protein has an imperfectcell wall binding domain (XPXTG) followed by a transmembrane spanningdomain encompassing residues 279-294. The emaB nucleotide sequence isnot homologous to known sequences of bacterial genes. The translatedamino acid sequence, however, shares segmental homology with a number ofcharacterized proteins, including a type 2 fimbrial structural subunitof Actinomyces naeslandii (28% homology over 222 amino acids), the T6protein of S. pyogenes (26% homology over 266 amino acids) (20), and aS. epidermidis putative cell-surface adhesin (24% identity over 197amino acids). The first of these proteins mediates adhesion of S. aureusto collagen and is postulated to contribute to the pathogenesis ofosteomyelifis and infectious arthritis.

[0363] emaC

[0364] The third ORF, emaC, begins 2 bp 3′ of emaB and is the sametranscriptional orientation. It is 918 bp long, with a predicted proteinproduct of 305 amino acids and Mr 34.5. The nucleic acid sequence (SEQID NO: 5) and predicted amino acid sequence (SEQ ID NO: 6) of emaC aredepicted in FIG. 4. The EmaC protein is a nonrepetitive protein. The 30amino acid N-terminus of the predicted protein is consistent with asignal peptide. The mature protein has a transmembrane spanning domainemcompassing residues 265-281. The emaC nucleotide sequence is nothomologous to known sequences of bacterial genes. The translated aminoacid sequence, however, shares segmental homology with a number ofcharacterized proteins, including proteins associated with the assemblyof type 2 fimbrial structural subunit of Actinomyces naeslandii (38%homology over 234 amino acids) (16). These proteins are required for theassembly of type 2 fimbria.

[0365] emaD

[0366] The fourth ORF, emaD, is 852 bp long, overlaps emaC by 47 bp, andis in the same transcriptional orientation. The predicted proteinproduct is 284 amino acids and Mr 33.1. The nucleic acid sequence (SEQID NO: 7) and predicted amino acid sequence (SEQ ID NO:8) of emaD areshown in FIG. 5. No indentifiable N-terminal signal sequence is presentand potential transmembrane segments are present at positions 19-35 and252-280. The mature protein is not repetative and lacks a cell wallbinding domain. The emaD nucleotide sequence is not homologous to knownsequences of bacterial genes. The translated amino acid sequence, sharessegmental homology with the same fimbria-associated proteins ofActinomyces as does EmaC.

[0367] emaE

[0368] The fifth ORF, emaE, begins 42 bp 3′ of emaD and is in the sametranscriptional orientation. It is 2712 bp long, with a predictedprotein product of 904 aa and Mr 100.9. FIG. 6 depicts the nucleic acidsequence (SEQ ID NO: 9) and predicted amino acid sequence (SEQ ID NO:10) of emaE. The predicted EmaE protein is a nonrepetitive protein. Anobvious N-terminal signal peptide is not evident but a putativetransmembrane region is located at residues 24-40. The mature proteinhas an imperfect cell wall binding domain (XPXTGG (SEQ ID NO: 21))followed by a transmembrane spanning domain emcompassing residues880-896. The emaE nucleotide sequence is not homologous to knownsequences of bacterial genes. The translated amino acid sequence,however, shares segmental homology with a number of characterizedproteins, including the F1 and F2 fibronectin binding proteins of S.pyogenes (31% homology over 207 amino acids) (18, 19). These proteinsmediate high affinity binding to fibronectin, and are important in theadhesion of S. pyogenes to respiratory cells.

[0369] The similarity of the protein products of the ema locus tophysiologically important adhesins and invasins of other bacterialspecies suggests that the Ema proteins have a role in facilitating theadhesion of GBS to extracellular matrix components and to cell surfacesand subsequent invasion of epithelial and endothelial cells, the initialsteps in the pathogenesis of infection.

[0370] Several lines of evidence suggest the members of the ema and thespb locus may have similar functions, but are likely to representdistinct classes of proteins. The ema and spb locus genes are each andall similar to physiologically important adhesions and invasions of thebacterial species, however, both Spb1 and Spb2 have prototypical grampositive cell-wall binding domains, whereas the members of the ema locushave an unusual motif, suggesting a distinct mechanism of cell surfaceanchoring. Second, the spb locus is restricted to virulent serotypeIII-3 strains of GBS, whereas the ema locus appears to be ubiquitous inall GBS serotypes. Third, spb1 and spb2 are more homologous to oneanother than to members of the ema locus and ema genes are more closelyhomologous to one another than to spb1 and spb2.

EXAMPLE 2 Biologic Characterization of Novel GBS Genes

[0371] Isogenic Mutant Bacterial Strains

[0372] To identify biologic activity of these novel GBS genes, isogenicmutant bacterial strains are created which are identical in all respectsexcept for the presence or absence of a particular gene. Deletionmutants are created by allelic replacement. The relevant gene, with100-300 bp of flanking sequences, is subcloned and modified by thedeletion of an intragenic portion of the coding sequence and, in somecases, the insertion of a kanamycin resistance gene. The mutant gene iscloned into the suicide vector pHY304 (kindly provided by Dr. CraigRubens), a broad host range plasmid containing a temperature sensitiveori, erythromycin resistance gene (erm^(TS)), and a pBS multiple cloningsite. The pHY304 vector is a derivative of the vector pWV01 (Framson, P.E. et al (1997) Applied Environ Microbiology 63:3539-3547). Plasmidscontaining mutant genes are electroporated into strain 874391 and singlecross-over mutants are selected by antibiotic resistance at 37° C. Theresulting antibiotic resistant colonies are subjected to a temperatureshift to 30° C. Integration of the plasmid is unstable at thispermissive temperature because there are two functional ori's on thechromosome. Excised plasmid is eliminated by growth on nonselectivemedia for many generations, then colonies are screened for the presenceof the mutant allele by erythromycin-sensitivity. Double-crossovermutants are stable and do not require maintenance under drug selection.The mutant genotype is confirmed by Southern blotting or PCRdemonstrating the appropriate deletion. The resulting mutants arescreened for the presence of gene expression by Northern and Westernblot analysis. The phenotype of the knockout mutants is then comparedwith that of the wild type strain 874391 by examining growth rate andcolony morphology, and the expression of β-hemolysin and CAMP factor.Surface protein expression is assessed by Western blot, using polygonalsera from rabbits immunized with whole, heat-killed type III GBS.

[0373] In Vitro Models

[0374] A. Adherence

[0375] Adhesion of GBS to host cells is a prerequisite for invasivedisease. Three different cell types have the potential to be importantin this process: i) adhesion to respiratory epithelial cells is likelyto facilitate most early onset neonatal infections, ii) adhesion togastrointestinal epithelial cells has been postulated to be important inthe pathogenesis of late onset neonatal infections, and iii) adhesion toendothelial cells is necessary for both endocarditis and otherendovascular infections, and is likely to be the initial event in GBSmeningitis. The ability of wild type and mutant strains to adhere toepithelial and endothelial cells is compared in adhesion assays.

[0376] Four different cell lines are used to investigate the role ofnovel GBS genes in adhesion. GBS adhere to and invade A549 humanalveolar epithelial carcinoma cells and surface proteins appear to playan important role in this process (8). GBS binding to A549 cells is usedas an in vitro model for respiratory colonization GBS also adhere toC2BBeL, a human intestinal epithelial cell line, which is used as amodel for gastrointestinal colonization, and to HeLa cervical epithelialcells, a model for genital colonization and maternal infection. Forendothelial adhesion, two cell lines are studied: freshly isolated humanumbilical vein endothelial (HUVE) cells; and an immortalized human brainmicrovascular endothelial cell line (BMEC). Adhesion assays areperformed as described by Tamura et al (9). Cell lines are grown toconfluence in 96-well tissue culture plates in recommended media.Monolayers are washed with PBS and fixed with 0.5% gluteraldehyde.Following blocking with 5% BSA in PBS, cells are inoculated with variousinocula of GBS, centrifuged for 10 minutes at 2000 rpm and incubated for1 hour at 4° C. Nonadherent bacteria are removed by washing three timeswith 5% nonfat dry milk in PBS and bound bacteria are then eluted andplated quantitatively.

[0377] B. Invasion

[0378] GBS adhere to and invade respiratory epithelium, endothelium andBMEC (8, 10, 11). The ability of wild type and isogenic mutant GBSstrains to invade the above epithelial and endothelial cells are testedas previously described (8, 10, 11). Assays that distinguish the abilityof GBS to invade eukaryotic cells versus adhere to cells capitalize onthe inability of penicillin and gentamicin to enter host cells, allowingquantification of intracellular bacteria after extracellular bacteriaare killed. GBS are grown to the desired growth phase in TH broth,washed twice with PBS and resuspended in tissue culture media containing10% fetal calf serum. Tissue culture monolayers grown to confluence in24-well plates are inoculated with varying inocula of GBS, centrifugedat 800×g and incubated at 37° C. in 5% CO₂ for 2-6 hours. Extracellularbacteria are removed by washing four times with PBS. Cells are thenincubated in fresh medium with 5 mg/ml penicillin and 100 mg/mlgentamicin for 2 hours. Media is then removed, monolayers washed, andcells lysed by treatment with 0.025% Triton X-100. Cell lysates aresonicated to disrupt bacterial chains and aliquots platedquantitatively.

[0379] C. Antibody to GBS Proteins

[0380] The ability of specific antibody to the novel GBS proteins topromote opsonophagocytic killing of GBS is tested (12). Rabbits areimmunized with recombinant or purified GBS proteins produced by standardtechniques. Rabbit antiserum of different dilutions (ranging from{fraction (1/50)} to {fraction (1/5,000)}) that has been exhaustivelyabsorbed with the relevant isogenic mutant strain at 4° C. will beincubated with GBS in the presence of human complement andpolymorphonuclear leukocytes (3×10⁶). Opsonophagocytic killing isexpressed as the log number of CFU surviving following 1 hour ofincubation subtracted from the log of the number of CFU at the zero timepoint. Killing of wild type strains is compared to that of isogenicmutants lacking novel GBS proteins.

[0381] In Vivo Models

[0382] The neonatal rat has been used by numerous laboratories as amodel of GBS infection because it closely mimics human neonatalinfection (13). The contribution of novel genes to the pathogenesis ofGBS infections is tested by comparing wild type and mutant in thissystem. Rat pups are inoculated by two routes. First, pups areinoculated intranasally to mimic the respiratory infection and sepsistypical of early onset GBS infection. Secondly, intraperitoneal orsubcutaneous inoculation reproduces the high grade bacteremia associatedwith GBS sepsis and that precedes GBS meningitis (14).

[0383] Rat pups are inoculated with varying doses of GBS strains andmortality is determined. The level of bacteremia is determined byquantitative blood cultures. Lung, liver, spleen and meningeal tissueare preserved for histologic examination.

[0384] The ability of antiserum to the GBS proteins to protect neonatalrats from GBS infection is tested (13). Newborn rats (<18 hours old)receive an intraperitoneal injection of 0.5 ml of undiluted rabbitantiserum, followed by the intraperitoneal inoculation of the equivalentof one LD50 unit of GBS (usually about 5000 bacteria) in PBS. Mortalityand morbidity are then determined.

[0385] Role of Novel GBS Proteins in Vaccines

[0386] Several surface proteins of GBS, including C and Rib areimmunogenic and protective against GBS infection in infant rodent models(25, 26). None of these proteins are present in all GBS strains (27).Furthermore, each of these proteins has a repetitive structure. Thephenotypic variability of these repetitive proteins allows escapemutants expressing variant forms to evade host immune systems and maylimit the effectiveness of these vaccines (28). It is notable that eachof the predicted proteins of the spb and ema loci do not have arepetitive structure and would not have this disadvantage.

[0387] The novel GBS proteins we describe here may be useful antigensfor a GBS vaccine. The data presented herein indicates these proteinshave a role in mediating adhesion to and invasion of GBS to humanepithelial cells, thus antibody against these antigens may prevent theseinitial steps in infection. It is highly desirable to develop a vaccinethat prevents colonization of pregnant women and other individuals atincreased risk of invasive GBS infection, as this would eliminate mostinfections. Our data suggests that antibody against Spb1 is effective inreducing colonization or infection following colonization with highlyvirulent strains of serotype III, and therefore this protein is aparticularly useful vaccine antigen. Members of the ema locus, unlikespb1 and spb2, are ubiquitous in GBS and therefore have a role in theprevention of infection by multiple serotypes of GBS. An optimal vaccineformulation includes combinations of these antigens.

[0388] Two strategies are used to design GBS vaccines using these novelproteins. First, purified recombinant or affinity-purified proteins areused as vaccine antigens, singly or in combination (25). Second, theseproteins are used as carrier proteins for capsular polysaccharide oroligosaccharide-based vaccines. GBS polysaccharides and oligosaccharidesare generally poorly immunogenic and fail to elicit significant memoryand booster responses (29). Conjugation of these polysaccharides oroligosaccharides to protein carriers increases immunogenicity. GBSpolysaccharide conjugated to tetanus toxoid, for example, has been usedto immunize pregnant women and results in high levels of maternal serumanti-polysaccharide antibody which may be transferred to the fetus inthe third trimester of pregnancy (30). Selection of appropriate carrierproteins is important for the development of polysaccharide-proteinvaccine formulations. For example, Haemophilus influenzae type b poly-or oligasaccharide conjugated to different protein carriers has variableimmunogenicity and elicits antibody with varying avidity (31, 32).Repeated immunization with the same carrier protein may also suppressimmune responses by competition for specific B cells (epitopicsuppression) or other mechanisms. This is of particular concern for thedevelopment of GBS vaccines since recently developed polyaccharide andoligosaccharide-protein conjugate vaccines against the bacteria H.influenzae, S. pneumoniae, and N. meningitidis all utilize a restrictednumber of carrier proteins (tetanus toxoid, CRM197, diptheria toxoid),increasing the number of exposures to these carriers an individual islikely to recieve. A “designer” vaccine, composed of a GBSpolysaccharide or oligosaccharide coupled to a GBS-specific carrierprotein, such as the novel GBS polypeptides, provided herein,particularly including Spb1, EmaC and EmaE, may be a preferablestrategy. The large size of certain of these novel GBS antigens may alsobe an advantage to traditional carrier proteins as increasing size isassociated with improved immunogenicity.

EXAMPLE 3 Ema Homologs in Streptococci and Other Bacteria

[0389] As noted above, the GBS Ema proteins share segmental homologywith certain characterized proteins from other bacterial species,including bacterial adhesion and invasion proteins. The segmentalhomolog is noted as in the range of 24-39%. In addition, the Emaproteins demonstrate some homology to one another. A comparison of theema genes shows that EmaA and EmaB are 47% homologous, however, due tothe difference in their predicted lengths it is necessary to insert gapsin the EmaA sequence in order to line them up. The two Ema proteinswhich are most similar in structure, EmaC and EmaD share 48.7% aminoacid homology to one another. EmaA/B, EmaC/D and EmaE are each ≦20%homologous to one another.

[0390] The ema sequences were used to search the unannotated microbialgenomes (Eubacteria). The predicted Ema proteins were searched againsttranslations in all six frames (tblast x) of finished and unfinishedunannotated microbial genomes available at the web site of the NationalCenter for Biotechnology Information (NCBI). Segmental amino acidhomolog was identified.

[0391] EmaA has some segmental homolog with S. pneumoniae, E. faecalis,B. anthiacis and C. diptheriae. Ema B has some segmental homolog with B.anthracis. EmaE has segmental homology to S. pyogenes and lesserhomology to B. anthracis. Significant homology was identified betweenthe GBS EmaC and EmaD and proteins in other bacterial species. EmaC hassignificant (55% identity over 149 amino acids) homology to a region ofthe S. pneumoniae chromosome and the S. pyogenes chromosome (47%identity over 150 amino acids). Lesser segmental homology was found toE. faecalis, S. equi, and C. diptheriae. EmaD has strong segmentalhomology (66% over 184 amino acids) to a region of the S. pneumoniaechromosome, and lesser segmental homology to C. diphtheriae and S.pyogenes.

[0392] We have identified two Ema homologs in S. pneumoniae. These S.pneumoniae homologs show homology to EmaC and EmaD and, like EmaC andEmaD, also demonstrate homology to fimbria-associated protein ofActinomyces. The encoding nucleic acid and predicted amino acid sequenceof the first S. pneumoniae EmaC/D homolog are provided in SEQ ID NOS: 24and 23, respectively. The genome region nucleic acid including the firsthomolog encoding sequence is provided in SEQ ID NO: 22. The nucleic acidand predicted amino acid sequence of the second S. pneumoniae EmaC/Dhomolog are provided in SEQ ID NOS: 27 and 26 respectively. The genomicregion nucleic acid of this second homolog is found in SEQ ID NO: 25.

[0393] An EmaC/D homolog has been identified in Enterococcus faecalis bysearch and analysis. The E. faecalis EmaC/D homolog predicted amino acidsequence is provided in SEQ ID NO: 29. The nucleic acid sequenceencoding this E. faecalis Ema homolog is provided in SEQ ID NO: 30. Thenucleic acid sequence of E. faecalis which genomic region encodes theEmaC/D homolog is provided in SEQ ID NO: 28.

[0394] We have also identified an EmaD homolog in Corynebacteriumdiptheriae. The predicted amino acid sequence of the C. diptheriae EmaDhomolog is provided in SEQ ID NO: 32. C. diptheriae nucleic acidsequence which encodes the homolog is found in SEQ D NO: 33. Thecorresponding genomic region sequence of C. diptheriae is provided inSEQ ID NO: 31.

[0395] A predicted EmaC/D homolog has been identified in S. pyogenes.The predicted partial amino acid sequence of this Ema homolog providedin SEQ ID NO: 37.

[0396] A region of amino acids TLLTCTPYMINS/THRLLVR/KG (SEQ ID NO: 34)is found in GBS EmaC, GBS EmaD, in both the EmaC/D homologs of S.pneumoniae, and in the E. faecalis Ema homolog. A similar sequenceTLVTCTPYGINTHRLLVTA (SEQ ID NO: 35) is also found in the C. diptheriaeEma homolog. The S. pyogenes predicted Ema homolog has a similarsequence TLVTCTPYGVNTKRLLVRG (SEQ ID NO: 36) as well.

[0397] The following is a list of the references referred to in thisExample section.

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[0429] 32 Schlesinger Y, Granoff D M and Group TVS. Avidity andbactericidal activity of antibody elicited by different Haemophilusinfluenzae type b conjugate vaccines. Journal of the American MedicalAssociation 267:1489-1494, 1992.

[0430] This invention may be embodied in other forms or carried out inother ways without departing from the spirit or essentialcharacteristics thereof. The present disclosure is therefore to beconsidered as in all aspects illustrate and not restrictive, the scopeof the invention being indicated by the appended claims, and all changeswhich come within the meaning and range of equivalency are intended tobe embraced therein.

[0431] Various references are cited throughout this Specification, eachof which is incorporated herein by reference in its entirety.

1 37 1 737 DNA Streptococcus agalactiae 1 atgacccttg ttaaaaatcaagatgctctt gataaagcta ctgcaaatac agatgatgcg 60 gcatttttgg aaattccagttgcatcaact attaatgaaa aagcagtttt aggaaaagca 120 attgaaaata cttttgaacttcaatatgac catactcctg ataaagctga caatccaaaa 180 ccatctaatc ctccaagaaaaccagaagtt catactggtg ggaaacgatt tgtaaagaaa 240 gactcaacag aaacacaaacactaggtggt gctgagtttg atttgttggc ttctgatggg 300 acagcagtaa aatggacagatgctcttatt aaagcgaata ctaataaaaa ctatattgct 360 ggagaagctg ttactgggcaaccaatcaaa ttgaaatcac atacagacgg tacgtttgag 420 attaaaggtt tggcttatgcagttgatgcg aatgcagagg gtacagcagt aacttacaaa 480 ttaaaagaaa caaaagcaccagaaggttat gtaatccctg ataaagaaat cgagtttaca 540 gtatcacaaa catcttataatacaaaacca actgacatca cggttgatag tgctgatgca 600 acacctgata caattaaaaacaacaaacgt ccttcaatcc ctaatactgg tggtattggt 660 acggctatct ttgtcgctatcggtgctgcg gtgatggctt ttgctgttaa ggggatgaag 720 cgtcgtacaa aagataa 737 2245 PRT Streptococcus agalactiae 2 Met Thr Leu Val Lys Asn Gln Asp AlaLeu Asp Lys Ala Thr Ala Asn 1 5 10 15 Thr Asp Asp Ala Ala Phe Leu GluIle Pro Val Ala Ser Thr Ile Asn 20 25 30 Glu Lys Ala Val Leu Gly Lys AlaIle Glu Asn Thr Phe Glu Leu Gln 35 40 45 Tyr Asp His Thr Pro Asp Lys AlaAsp Asn Pro Lys Pro Ser Asn Pro 50 55 60 Pro Arg Lys Pro Glu Val His ThrGly Gly Lys Arg Phe Val Lys Lys 65 70 75 80 Asp Ser Thr Glu Thr Gln ThrLeu Gly Gly Ala Glu Phe Asp Leu Leu 85 90 95 Ala Ser Asp Gly Thr Ala ValLys Trp Thr Asp Ala Leu Ile Lys Ala 100 105 110 Asn Thr Asn Lys Asn TyrIle Ala Gly Glu Ala Val Thr Gly Gln Pro 115 120 125 Ile Lys Leu Lys SerHis Thr Asp Gly Thr Phe Glu Ile Lys Gly Leu 130 135 140 Ala Tyr Ala ValAsp Ala Asn Ala Glu Gly Thr Ala Val Thr Tyr Lys 145 150 155 160 Leu LysGlu Thr Lys Ala Pro Glu Gly Tyr Val Ile Pro Asp Lys Glu 165 170 175 IleGlu Phe Thr Val Ser Gln Thr Ser Tyr Asn Thr Lys Pro Thr Asp 180 185 190Ile Thr Val Asp Ser Ala Asp Ala Thr Pro Asp Thr Ile Lys Asn Asn 195 200205 Lys Arg Pro Ser Ile Pro Asn Thr Gly Gly Ile Gly Thr Ala Ile Phe 210215 220 Val Ala Ile Gly Ala Ala Val Met Ala Phe Ala Val Lys Gly Met Lys225 230 235 240 Arg Arg Thr Lys Asp 245 3 924 DNA Streptococcusagalactiae 3 atgaaacaaa cattaaaact tatgttttct tttctgttga tgttagggactatgtttgga 60 attagccaaa ctgttttagc gcaagaaact catcagttga cgattgttcatcttgaagca 120 agggatattg atcgtccaaa tccacagttg gagattgccc ctaaagaagggactccaatt 180 gaaggagtac tctatcagtt gtaccaatta aaatcaactg aagatggcgatttgttggca 240 cattggaatt ccctaactat cacagaattg aaaaaacagg cgcagcaggtttttgaagcc 300 actactaatc aacaaggaaa ggctacattt aaccaactac cagatggaatttattatggt 360 ctggcggtta aagccggtga aaaaaatcgt aatgtctcag ctttcttggttgacttgtct 420 gaggataaag tgatttatcc taaaatcatc tggtccacag gtgagttggacttgcttaaa 480 gttggtgtgg atggtgatac caaaaaacca ctagcaggcg ttgtctttgaactttatgaa 540 aagaatggta ggactcctat tcgtgtgaaa aatggggtgc attctcaagatattgacgct 600 gcaaaacatt tagaaacaga ttcatcaggg catatcagaa tttccgggctcatccatggg 660 gactatgtct taaaagaaat cgagacacag tcaggatatc agatcggacaggcagagact 720 gctgtgacta ttgaaaaatc aaaaacagta acagtaacga ttgaaaataaaaaagttccg 780 acacctaaag tgccatctcg aggaggtctt attcccaaaa caggtgagcaacaggcaatg 840 gcacttgtaa ttattggtgg tattttaatt gctttagcct tacgattactatcaaaacat 900 cggaaacatc aaaataagga ttag 924 4 307 PRT Streptococcusagalactiae 4 Met Lys Gln Thr Leu Lys Leu Met Phe Ser Phe Leu Leu Met LeuGly 1 5 10 15 Thr Met Phe Gly Ile Ser Gln Thr Val Leu Ala Gln Glu ThrHis Gln 20 25 30 Leu Thr Ile Val His Leu Glu Ala Arg Asp Ile Asp Arg ProAsn Pro 35 40 45 Gln Leu Glu Ile Ala Pro Lys Glu Gly Thr Pro Ile Glu GlyVal Leu 50 55 60 Tyr Gln Leu Tyr Gln Leu Lys Ser Thr Glu Asp Gly Asp LeuLeu Ala 65 70 75 80 His Trp Asn Ser Leu Thr Ile Thr Glu Leu Lys Lys GlnAla Gln Gln 85 90 95 Val Phe Glu Ala Thr Thr Asn Gln Gln Gly Lys Ala ThrPhe Asn Gln 100 105 110 Leu Pro Asp Gly Ile Tyr Tyr Gly Leu Ala Val LysAla Gly Glu Lys 115 120 125 Asn Arg Asn Val Ser Ala Phe Leu Val Asp LeuSer Glu Asp Lys Val 130 135 140 Ile Tyr Pro Lys Ile Ile Trp Ser Thr GlyGlu Leu Asp Leu Leu Lys 145 150 155 160 Val Gly Val Asp Gly Asp Thr LysLys Pro Leu Ala Gly Val Val Phe 165 170 175 Glu Leu Tyr Glu Lys Asn GlyArg Thr Pro Ile Arg Val Lys Asn Gly 180 185 190 Val His Ser Gln Asp IleAsp Ala Ala Lys His Leu Glu Thr Asp Ser 195 200 205 Ser Gly His Ile ArgIle Ser Gly Leu Ile His Gly Asp Tyr Val Leu 210 215 220 Lys Glu Ile GluThr Gln Ser Gly Tyr Gln Ile Gly Gln Ala Glu Thr 225 230 235 240 Ala ValThr Ile Glu Lys Ser Lys Thr Val Thr Val Thr Ile Glu Asn 245 250 255 LysLys Val Pro Thr Pro Lys Val Pro Ser Arg Gly Gly Leu Ile Pro 260 265 270Lys Thr Gly Glu Gln Gln Ala Met Ala Leu Val Ile Ile Gly Gly Ile 275 280285 Leu Ile Ala Leu Ala Leu Arg Leu Leu Ser Lys His Arg Lys His Gln 290295 300 Asn Lys Asp 305 5 918 DNA Streptococcus agalactiae 5 atgggacaaaaatcaaaaat atctctagct acgaatattc gtatatggat ttttcgttta 60 attttcttagcgggtttcct tgttttggca tttcccatcg ttagtcaggt catgtacttt 120 caagcctctcacgccaatat taatgctttt aaagaagctg ttaccaagat tgaccgggtg 180 gagattaatcggcgtttaga acttgcttat gcttataacg ccagtatagc aggtgccaaa 240 actaatggcgaatatccagc gcttaaagac ccctactctg ctgaacaaaa gcaggcaggg 300 gtcgttgagtacgcccgcat gcttgaagtc aaagaacaaa taggtcatgt gattattcca 360 agaattaatcaggatatccc tatttacgct ggctctgctg aagaaaatct tcagaggggc 420 gttggacatttagaggggac cagtcttcca gtcggtggtg agtcaactca tgccgttcta 480 actgcccatcgagggctacc aacggccaag ctatttacca atttagacaa ggtaacagta 540 ggtgaccgtttttacattga acacatcggc ggaaagattg cttatcaggt agaccaaatc 600 aaagttatcgcccctgatca gttagaggat ttgtacgtga ttcaaggaga agatcacgtc 660 accctattaacttgcacacc ttatatgata aatagtcatc gcctcctcgt tcgaggcaag 720 cgaattccttatgtggaaaa aacagtgcag aaagattcaa agaccttcag gcaacaacaa 780 tacctaacctatgctatgtg ggtagtcgtt ggacttatct tgctgtcgct tctcatttgg 840 tttaaaaagacgaaacagaa aaagcggaga aagaatgaaa aagcggctag tcaaaatagt 900 cacaataattcgaaataa 918 6 305 PRT Streptococcus agalactiae 6 Met Gly Gln Lys SerLys Ile Ser Leu Ala Thr Asn Ile Arg Ile Trp 1 5 10 15 Ile Phe Arg LeuIle Phe Leu Ala Gly Phe Leu Val Leu Ala Phe Pro 20 25 30 Ile Val Ser GlnVal Met Tyr Phe Gln Ala Ser His Ala Asn Ile Asn 35 40 45 Ala Phe Lys GluAla Val Thr Lys Ile Asp Arg Val Glu Ile Asn Arg 50 55 60 Arg Leu Glu LeuAla Tyr Ala Tyr Asn Ala Ser Ile Ala Gly Ala Lys 65 70 75 80 Thr Asn GlyGlu Tyr Pro Ala Leu Lys Asp Pro Tyr Ser Ala Glu Gln 85 90 95 Lys Gln AlaGly Val Val Glu Tyr Ala Arg Met Leu Glu Val Lys Glu 100 105 110 Gln IleGly His Val Ile Ile Pro Arg Ile Asn Gln Asp Ile Pro Ile 115 120 125 TyrAla Gly Ser Ala Glu Glu Asn Leu Gln Arg Gly Val Gly His Leu 130 135 140Glu Gly Thr Ser Leu Pro Val Gly Gly Glu Ser Thr His Ala Val Leu 145 150155 160 Thr Ala His Arg Gly Leu Pro Thr Ala Lys Leu Phe Thr Asn Leu Asp165 170 175 Lys Val Thr Val Gly Asp Arg Phe Tyr Ile Glu His Ile Gly GlyLys 180 185 190 Ile Ala Tyr Gln Val Asp Gln Ile Lys Val Ile Ala Pro AspGln Leu 195 200 205 Glu Asp Leu Tyr Val Ile Gln Gly Glu Asp His Val ThrLeu Leu Thr 210 215 220 Cys Thr Pro Tyr Met Ile Asn Ser His Arg Leu LeuVal Arg Gly Lys 225 230 235 240 Arg Ile Pro Tyr Val Glu Lys Thr Val GlnLys Asp Ser Lys Thr Phe 245 250 255 Arg Gln Gln Gln Tyr Leu Thr Tyr AlaMet Trp Val Val Val Gly Leu 260 265 270 Ile Leu Leu Ser Leu Leu Ile TrpPhe Lys Lys Thr Lys Gln Lys Lys 275 280 285 Arg Arg Lys Asn Glu Lys AlaAla Ser Gln Asn Ser His Asn Asn Ser 290 295 300 Lys 305 7 852 DNAStreptococcus agalactiae 7 atgaaaaagc ggctagtcaa aatagtcaca ataattcgaaataataaaat cagaaccctc 60 atttttgtga tgggaagtct gattctctta tttccgattgtgagccaggt aagttactac 120 cttgcttcgc atcaaaatat taatcaattt aagcgggaagtcgctaagat tgatactaat 180 acggttgaac gacgcatcgc tttagctaat gcttacaatgagacgttatc aaggaatccc 240 ttgcttatag acccttttac cagtaagcaa aaagaaggtttgagagagta tgctcgtatg 300 cttgaagttc atgagcaaat aggtcatgtg gcaatcccaagtatttgggt tgatattcca 360 atttatgctg gaacatccga aactgtgctt cagaaaggtagtgggcattt ggagggaacc 420 agtcttccag tgggaggttt gtcaacccat tcagtactaactgcccaccg tggcttgcca 480 acagctaggc tatttaccga cttaaataaa gttaaaaaaggccagatttt ctatgtgacg 540 aacatcaagg aaacacttgc ctacaaagtc gtgtctatcaaagttgtgga tccaacagct 600 ttaagtgagg ttaagattgt caatggtaag gattatataaccttgctgac ttgcacacct 660 tacatgatca atagtcatcg tctcttggta aaaggagagcgtattcctta tgattctacc 720 gaggcggaaa agcacaaaga acaaaccgta caagattatcgtttgtcact agtgttgaag 780 atactactag tattattaat tggactcttc atcgtgataatgatgagaag atggatgcaa 840 catcgtcaat aa 852 8 283 PRT Streptococcusagalactiae 8 Met Lys Lys Arg Leu Val Lys Ile Val Thr Ile Ile Arg Asn AsnLys 1 5 10 15 Ile Arg Thr Leu Ile Phe Val Met Gly Ser Leu Ile Leu LeuPhe Pro 20 25 30 Ile Val Ser Gln Val Ser Tyr Tyr Leu Ala Ser His Gln AsnIle Asn 35 40 45 Gln Phe Lys Arg Glu Val Ala Lys Ile Asp Thr Asn Thr ValGlu Arg 50 55 60 Arg Ile Ala Leu Ala Asn Ala Tyr Asn Glu Thr Leu Ser ArgAsn Pro 65 70 75 80 Leu Leu Ile Asp Pro Phe Thr Ser Lys Gln Lys Glu GlyLeu Arg Glu 85 90 95 Tyr Ala Arg Met Leu Glu Val His Glu Gln Ile Gly HisVal Ala Ile 100 105 110 Pro Ser Ile Gly Val Asp Ile Pro Ile Tyr Ala GlyThr Ser Glu Thr 115 120 125 Val Leu Gln Lys Gly Ser Gly His Leu Glu GlyThr Ser Leu Pro Val 130 135 140 Gly Gly Leu Ser Thr His Ser Val Leu ThrAla His Arg Gly Leu Pro 145 150 155 160 Thr Ala Arg Leu Phe Thr Asp LeuAsn Lys Val Lys Lys Gly Gln Ile 165 170 175 Phe Tyr Val Thr Asn Ile LysGlu Thr Leu Ala Tyr Lys Val Val Ser 180 185 190 Ile Lys Val Val Asp ProThr Ala Leu Ser Glu Val Lys Ile Val Asn 195 200 205 Gly Lys Asp Tyr IleThr Leu Leu Thr Cys Thr Pro Tyr Met Ile Asn 210 215 220 Ser His Arg LeuLeu Val Lys Gly Glu Arg Ile Pro Tyr Asp Ser Thr 225 230 235 240 Glu AlaGlu Lys His Lys Glu Gln Thr Val Gln Asp Tyr Arg Leu Ser 245 250 255 LeuVal Leu Lys Ile Leu Leu Val Leu Leu Ile Gly Leu Phe Ile Val 260 265 270Ile Met Met Arg Arg Trp Met Gln His Arg Gln 275 280 9 2712 DNAStreptococcus agalactiae 9 atgatgattg tgaataatgg ttatctagaa gggagaaaaatgaaaaagag acaaaaaata 60 tggagagggt tatcagttac tttactaatc ctgtcccaaattccatttgg tatattggta 120 caaggtgaaa cccaagatac caatcaagca cttggaaaagtaattgttaa aaaaacggga 180 gacaatgcta caccattagg caaagcgact tttgtgttaaaaaatgacaa tgataagtca 240 gaaacaagtc acgaaacggt agagggttct ggagaagcaacctttgaaaa cataaaacct 300 ggagactaca cattaagaga agaaacagca ccaattggttataaaaaaac tgataaaacc 360 tggaaagtta aagttgcaga taacggagca acaataatcgagggtatgga tgcagataaa 420 gcagagaaac gaaaagaagt tttgaatgcc caatatccaaaatcagctat ttatgaggat 480 acaaaagaaa attacccatt agttaatgta gagggttccaaagttggtga acaatacaaa 540 gcattgaatc caataaatgg aaaagatggt cgaagagagattgctgaagg ttggttatca 600 aaaaaaaata caggggtcaa tgatctcgat aagaataaatataaaattga attaactgtt 660 gagggtaaaa ccactgttga aacgaaagaa cttaatcaaccactagatgt cgttgtgcta 720 ttagataatt caaatagtat gaataatgaa agagccaataattctcaaag agcattaaaa 780 gctggggaag cagttgaaaa gctgattgat aaaattacatcaaataaaga caatagagta 840 gctcttgtga catatgcctc aaccattttt gatggtactgaagcgaccgt atcaaaggga 900 gttgccgatc aaaatggtaa agcgctgaat gatagtgtatcatgggatta tcataaaact 960 acttttacag caactacaca taattacagt tatttaaatttaacaaatga tgctaacgaa 1020 gttaatattc taaagtcaag aattccaaag gaagcggagcatataaatgg ggatcgcacg 1080 ctctatcaat ttggtgcgac atttactcaa aaagctctaatgaaagcaaa tgaaatttta 1140 gagacacaaa gttctaatgc tagaaaaaaa cttatttttcacgtaactga tggtgtccct 1200 acgatgtctt atgccataaa ttttaatcct tatatatcaacatcttacca aaaccagttt 1260 aattcttttt taaataaaat accagataga agtggtattctccaagagga ttttataatc 1320 aatggtgatg attatcaaat agtaaaagga gatggagagagttttaaact gttttcggat 1380 agaaaagttc ctgttactgg aggaacgaca caagcagcttatcgagtacc gcaaaatcaa 1440 ctctctgtaa tgagtaatga gggatatgca attaatagtggatatattta tctctattgg 1500 agagattaca actgggtcta tccatttgat cctaagacaaagaaagtttc tgcaacgaaa 1560 caaatcaaaa ctcatggtga gccaacaaca ttatactttaatggaaatat aagacctaaa 1620 ggttatgaca tttttactgt tgggattggt gtaaacggagatcctggtgc aactcctctt 1680 gaagctgaga aatttatgca atcaatatca agtaaaacagaaaattatac taatgttgat 1740 gatacaaata aaatttatga tgagctaaat aaatactttaaaacaattgt tgaggaaaaa 1800 cattctattg ttgatggaaa tgtgactgat cctatgggagagatgattga attccaatta 1860 aaaaatggtc aaagttttac acatgatgat tacgttttggttggaaatga tggcagtcaa 1920 ttaaaaaatg gtgtggctct tggtggacca aacagtgatgggggaatttt aaaagatgtt 1980 acagtgactt atgataagac atctcaaacc atcaaaatcaatcatttgaa cttaggaagt 2040 ggacaaaaag tagttcttac ctatgatgta cgtttaaaagataactatat aagtaacaaa 2100 ttttacaata caaataatcg tacaacgcta agtccgaagagtgaaaaaga accaaatact 2160 attcgtgatt tcccaattcc caaaattcgt gatgttcgtgagtttccggt actaaccatc 2220 agtaatcaga agaaaatggg tgaggttgaa tttattaaagttaataaaga caaacattca 2280 gaatcgcttt tgggagctaa gtttcaactt cagatagaaaaagatttttc tgggtataag 2340 caatttgttc cagagggaag tgatgttaca acaaagaatgatggtaaaat ttattttaaa 2400 gcacttcaag atggtaacta taaattatat gaaatttcaagtccagatgg ctatatagag 2460 gttaaaacga aacctgttgt gacatttaca attcaaaatggagaagttac gaacctgaaa 2520 gcagatccaa atgctaataa aaatcaaatc gggtatcttgaaggaaatgg taaacatctt 2580 attaccaaca ctcccaaacg cccaccaggt gtttttcctaaaacaggggg aattggtaca 2640 attgtctata tattagttgg ttctactttt atgatacttaccatttgttc tttccgtcgt 2700 aaacaattgt aa 2712 10 903 PRT Streptococcusagalactiae 10 Met Met Ile Val Asn Asn Gly Tyr Leu Glu Gly Arg Lys MetLys Lys 1 5 10 15 Arg Gln Lys Ile Trp Arg Gly Leu Ser Val Thr Leu LeuIle Leu Ser 20 25 30 Gln Ile Pro Phe Gly Ile Leu Val Gln Gly Glu Thr GlnAsp Thr Asn 35 40 45 Gln Ala Leu Gly Lys Val Ile Val Lys Lys Thr Gly AspAsn Ala Thr 50 55 60 Pro Leu Gly Lys Ala Thr Phe Val Leu Lys Asn Asp AsnAsp Lys Ser 65 70 75 80 Glu Thr Ser His Glu Thr Val Glu Gly Ser Gly GluAla Thr Phe Glu 85 90 95 Asn Ile Lys Pro Gly Asp Tyr Thr Leu Arg Glu GluThr Ala Pro Ile 100 105 110 Gly Tyr Lys Lys Thr Asp Lys Thr Trp Lys ValLys Val Ala Asp Asn 115 120 125 Gly Ala Thr Ile Ile Glu Gly Met Asp AlaAsp Lys Ala Glu Lys Arg 130 135 140 Lys Glu Val Leu Asn Ala Gln Tyr ProLys Ser Ala Ile Tyr Glu Asp 145 150 155 160 Thr Lys Glu Asn Tyr Pro LeuVal Asn Val Glu Gly Ser Lys Val Gly 165 170 175 Glu Gln Tyr Lys Ala LeuAsn Pro Ile Asn Gly Lys Asp Gly Arg Arg 180 185 190 Glu Ile Ala Glu GlyTrp Leu Ser Lys Lys Asn Thr Gly Val Asn Asp 195 200 205 Leu Asp Lys AsnLys Tyr Lys Ile Glu Leu Thr Val Glu Gly Lys Thr 210 215 220 Thr Val GluThr Lys Glu Leu Asn Gln Pro Leu Asp Val Val Val Leu 225 230 235 240 LeuAsp Asn Ser Asn Ser Met Asn Asn Glu Arg Ala Asn Asn Ser Gln 245 250 255Arg Ala Leu Lys Ala Gly Glu Ala Val Glu Lys Leu Ile Asp Lys Ile 260 265270 Thr Ser Asn Lys Asp Asn Arg Val Ala Leu Val Thr Tyr Ala Ser Thr 275280 285 Ile Phe Asp Gly Thr Glu Ala Thr Val Ser Lys Gly Val Ala Asp Gln290 295 300 Asn Gly Lys Ala Leu Asn Asp Ser Val Ser Trp Asp Tyr His LysThr 305 310 315 320 Thr Phe Thr Ala Thr Thr His Asn Tyr Ser Tyr Leu AsnLeu Thr Asn 325 330 335 Asp Ala Asn Glu Val Asn Ile Leu Lys Ser Arg IlePro Lys Glu Ala 340 345 350 Glu His Ile Asn Gly Asp Arg Thr Leu Tyr GlnPhe Gly Ala Thr Phe 355 360 365 Thr Gln Lys Ala Leu Met Lys Ala Asn GluIle Leu Glu Thr Gln Ser 370 375 380 Ser Asn Ala Arg Lys Lys Leu Ile PheHis Val Thr Asp Gly Val Pro 385 390 395 400 Thr Met Ser Tyr Ala Ile AsnPhe Asn Pro Tyr Ile Ser Thr Ser Tyr 405 410 415 Gln Asn Gln Phe Asn SerPhe Leu Asn Lys Ile Pro Asp Arg Ser Gly 420 425 430 Ile Leu Gln Glu AspPhe Ile Ile Asn Gly Asp Asp Tyr Gln Ile Val 435 440 445 Lys Gly Asp GlyGlu Ser Phe Lys Leu Phe Ser Asp Arg Lys Val Pro 450 455 460 Val Thr GlyGly Thr Thr Gln Ala Ala Tyr Arg Val Pro Gln Asn Gln 465 470 475 480 LeuSer Val Met Ser Asn Glu Gly Tyr Ala Ile Asn Ser Gly Tyr Ile 485 490 495Tyr Leu Tyr Trp Arg Asp Tyr Asn Trp Val Tyr Pro Phe Asp Pro Lys 500 505510 Thr Lys Lys Val Ser Ala Thr Lys Gln Ile Lys Thr His Gly Glu Pro 515520 525 Thr Thr Leu Tyr Phe Asn Gly Asn Ile Arg Pro Lys Gly Tyr Asp Ile530 535 540 Phe Thr Val Gly Ile Gly Val Asn Gly Asp Pro Gly Ala Thr ProLeu 545 550 555 560 Glu Ala Glu Lys Phe Met Gln Ser Ile Ser Ser Lys ThrGlu Asn Tyr 565 570 575 Thr Asn Val Asp Asp Thr Asn Lys Ile Tyr Asp GluLeu Asn Lys Tyr 580 585 590 Phe Lys Thr Ile Val Glu Glu Lys His Ser IleVal Asp Gly Asn Val 595 600 605 Thr Asp Pro Met Gly Glu Met Ile Glu PheGln Leu Lys Asn Gly Gln 610 615 620 Ser Phe Thr His Asp Asp Tyr Val LeuVal Gly Asn Asp Gly Ser Gln 625 630 635 640 Leu Lys Asn Gly Val Ala LeuGly Gly Pro Asn Ser Asp Gly Gly Ile 645 650 655 Leu Lys Asp Val Thr ValThr Tyr Asp Lys Thr Ser Gln Thr Ile Lys 660 665 670 Ile Asn His Leu AsnLeu Gly Ser Gly Gln Lys Val Val Leu Thr Tyr 675 680 685 Asp Val Arg LeuLys Asp Asn Tyr Ile Ser Asn Lys Phe Tyr Asn Thr 690 695 700 Asn Asn ArgThr Thr Leu Ser Pro Lys Ser Glu Lys Glu Pro Asn Thr 705 710 715 720 IleArg Asp Phe Pro Ile Pro Lys Ile Arg Asp Val Arg Glu Phe Pro 725 730 735Val Leu Thr Ile Ser Asn Gln Lys Lys Met Gly Glu Val Glu Phe Ile 740 745750 Lys Val Asn Lys Asp Lys His Ser Glu Ser Leu Leu Gly Ala Lys Phe 755760 765 Gln Leu Gln Ile Glu Lys Asp Phe Ser Gly Tyr Lys Gln Phe Val Pro770 775 780 Glu Gly Ser Asp Val Thr Thr Lys Asn Asp Gly Lys Ile Tyr PheLys 785 790 795 800 Ala Leu Gln Asp Gly Asn Tyr Lys Leu Tyr Glu Ile SerSer Pro Asp 805 810 815 Gly Tyr Ile Glu Val Lys Thr Lys Pro Val Val ThrPhe Thr Ile Gln 820 825 830 Asn Gly Glu Val Thr Asn Leu Lys Ala Asp ProAsn Ala Asn Lys Asn 835 840 845 Gln Ile Gly Tyr Leu Glu Gly Asn Gly LysHis Leu Ile Thr Asn Thr 850 855 860 Pro Lys Arg Pro Pro Gly Val Phe ProLys Thr Gly Gly Ile Gly Thr 865 870 875 880 Ile Val Tyr Ile Leu Val GlySer Thr Phe Met Ile Leu Thr Ile Cys 885 890 895 Ser Phe Arg Arg Lys GlnLeu 900 11 21 DNA Artificial sequence oligonucleotide 11 ctaggtggatccttcggcaa t 21 12 10 DNA Artificial sequence oligonucleotide 12cgattgccga 10 13 25 DNA Artificial sequence oligonucleotide 13aggcaactgt gctaaccgag ggaat 25 14 11 DNA Artificial sequenceoligonucleotide 14 cgattccctc g 11 15 1509 DNA Streptococcus agalactiae15 atgaaaaaga aaatgattca atcgctgtta gtggcgagtt tagcatttgg tatggctgta 60tcaccagtta cgccgatagc ttttgccgct gagacaggga caattacagt tcaagatact 120caaaaaggcg caacctataa agcatataaa gtttttgatg cagaaataga taatgcaaat 180gtatctgatt cgaataaaga tggagcttct tatttaattc ctcaaggtaa agaagctgag 240tataaagctt caactgattt taattctctt tttacgacaa ctactaatgg agggagaaca 300tatgtaacta aaaaagatac tgcgtcagca aatgagattg cgacatgggc taaatctata 360tcagctaata ctacaccagt ttccactgtt actgagtcaa ataatgatgg tactgaggtt 420attaatgttt cccaatatgg atattattat gtttctagca ctgttaataa tggagctgta 480attatggtta catctgtaac tccaaatgct actattcatg aaaagaatac tgatgcgaca 540tggggagatg gtggtggaaa aactgtagat caaaaaacgt actcggttgg tgatacagtc 600aaatatacta ttacttataa gaatgcagtc aattatcatg gtacagaaaa agtgtatcaa 660tatgttataa aggatactat gccatctgct tctgtagttg atttgaacga agggtcttat 720gaagtaacta ttactgatgg atcagggaat attacaactc taactcaagg ttcggaaaaa 780gcaactggga agtataacct gttagaggaa aataataatt tcacgattac tattccgtgg 840gcagctacca atactccaac cggaaatact caaaatggag ctaatgatga ctttttttat 900aagggaataa atacaatcac agtcacttat acaggagtat taaagagtgg agctaaacca 960ggttcagctg atttaccaga aaatacaaac attgcgacca tcaaccccaa tactagcaat 1020gatgacccag gtcaaaaagt aacagtgagg gatggtcaaa ttactataaa aaaaattgat 1080ggttccacaa aagcttcatt acaaggtgct atatttgttt taaagaatgc tacgggtcaa 1140tttctaaact ttaacgatac aaataacgtt gaatggggca cagaagctaa tgcaacagaa 1200tatacaacag gagcagatgg tataattacc attacaggct tgaaagaagg tacatactat 1260ctagttgaga aaaaggctcc cttaggttac aatttgttag ataactctca gaaggttatt 1320ttaggagatg gagccactga tacgactaat tcagataacc ttttagttaa cccaactgtt 1380gaaaataaca aaggtactga gttgccttca acaggtggta ttggtacaac aattttctac 1440attataggtg caattttagt aataggagca ggtatcgtgc ttgttgctcg tcgtcgttta 1500cgttcttaa 1509 16 502 PRT Streptococcus agalactiae 16 Met Lys Lys LysMet Ile Gln Ser Leu Leu Val Ala Ser Leu Ala Phe 1 5 10 15 Gly Met AlaVal Ser Pro Val Thr Pro Ile Ala Phe Ala Ala Glu Thr 20 25 30 Gly Thr IleThr Val Gln Asp Thr Gln Lys Gly Ala Thr Tyr Lys Ala 35 40 45 Tyr Lys ValPhe Asp Ala Glu Ile Asp Asn Ala Asn Val Ser Asp Ser 50 55 60 Asn Lys AspGly Ala Ser Tyr Leu Ile Pro Gln Gly Lys Glu Ala Glu 65 70 75 80 Tyr LysAla Ser Thr Asp Phe Asn Ser Leu Phe Thr Thr Thr Thr Asn 85 90 95 Gly GlyArg Thr Tyr Val Thr Lys Lys Asp Thr Ala Ser Ala Asn Glu 100 105 110 IleAla Thr Trp Ala Lys Ser Ile Ser Ala Asn Thr Thr Pro Val Ser 115 120 125Thr Val Thr Glu Ser Asn Asn Asp Gly Thr Glu Val Ile Asn Val Ser 130 135140 Gln Tyr Gly Tyr Tyr Tyr Val Ser Ser Thr Val Asn Asn Gly Ala Val 145150 155 160 Ile Met Val Thr Ser Val Thr Pro Asn Ala Thr Ile His Glu LysAsn 165 170 175 Thr Asp Ala Thr Trp Gly Asp Gly Gly Gly Lys Thr Val AspGln Lys 180 185 190 Thr Tyr Ser Val Gly Asp Thr Val Lys Tyr Thr Ile ThrTyr Lys Asn 195 200 205 Ala Val Asn Tyr His Gly Thr Glu Lys Val Tyr GlnTyr Val Ile Lys 210 215 220 Asp Thr Met Pro Ser Ala Ser Val Val Asp LeuAsn Glu Gly Ser Tyr 225 230 235 240 Glu Val Thr Ile Thr Asp Gly Ser GlyAsn Ile Thr Thr Leu Thr Gln 245 250 255 Gly Ser Glu Lys Ala Thr Gly LysTyr Asn Leu Leu Glu Glu Asn Asn 260 265 270 Asn Phe Thr Ile Thr Ile ProTrp Ala Ala Thr Asn Thr Pro Thr Gly 275 280 285 Asn Thr Gln Asn Gly AlaAsn Asp Asp Phe Phe Tyr Lys Gly Ile Asn 290 295 300 Thr Ile Thr Val ThrTyr Thr Gly Val Leu Lys Ser Gly Ala Lys Pro 305 310 315 320 Gly Ser AlaAsp Leu Pro Glu Asn Thr Asn Ile Ala Thr Ile Asn Pro 325 330 335 Asn ThrSer Asn Asp Asp Pro Gly Gln Lys Val Thr Val Arg Asp Gly 340 345 350 GlnIle Thr Ile Lys Lys Ile Asp Gly Ser Thr Lys Ala Ser Leu Gln 355 360 365Gly Ala Ile Phe Val Leu Lys Asn Ala Thr Gly Gln Phe Leu Asn Phe 370 375380 Asn Asp Thr Asn Asn Val Glu Trp Gly Thr Glu Ala Asn Ala Thr Glu 385390 395 400 Tyr Thr Thr Gly Ala Asp Gly Ile Ile Thr Ile Thr Gly Leu LysGlu 405 410 415 Gly Thr Tyr Tyr Leu Val Glu Lys Lys Ala Pro Leu Gly TyrAsn Leu 420 425 430 Leu Asp Asn Ser Gln Lys Val Ile Leu Gly Asp Gly AlaThr Asp Thr 435 440 445 Thr Asn Ser Asp Asn Leu Leu Val Asn Pro Thr ValGlu Asn Asn Lys 450 455 460 Gly Thr Glu Leu Pro Ser Thr Gly Gly Ile GlyThr Thr Ile Phe Tyr 465 470 475 480 Ile Ile Gly Ala Ile Leu Val Ile GlyAla Gly Ile Val Leu Val Ala 485 490 495 Arg Arg Arg Leu Arg Ser 500 17 5PRT Artificial sequence consensus 17 Leu Pro Xaa Thr Gly 1 5 18 1683 DNAStreptococcus agalactiae 18 atggtgatcg tattccggat tatacagata ttacaagggattatatccaa gatccttcag 60 gtacatatta ttataagtat gattcacgag ataaagatcccgactcaact aaagatgcct 120 attatacgac agatactagt ctcatcaaat gttgatacaacaactaagta caagtacgta 180 aaagacgctt acaaattagt cggttggtat tatgttaatccatatggtag tattagacct 240 tataactttt caggtgctgt aactcaagat atcaatttaagagctatttg gcgaaaggct 300 ggagattatc atattatata cagcaatgat gctgttggtacagatggaaa gccagcattg 360 gatgcttctg gtcagcaatt acaaacaagt aatgagcctactgaccctga ttcctatgac 420 gatggctccc attcagcctt actgagacgt ccgacaatgccagatggcta tcgtttccgt 480 ggctggtggt acaatggtaa aatttataac ccatatgattccattgatat tgacgcccat 540 ttagcagatg ctaataaaaa tatcaccata aaacctgtcattattccagt aggagatatc 600 aaattagaag atacctccat caaatacaat ggtaacggtggtactagagt agaaaatggt 660 aatgtggtaa cacaagtgga gacaccgcgt atggagttgaatagcacaac tacaattcct 720 gaaaaccaat actttacaag gacaggttac aaccttattggttggcatca tgataaggat 780 ttagctgata caggacgtgt ggaatttaca gcaggtcaatcaataggtat tgataacaac 840 cttgatgcaa caaatacctt atatgctgtt tggcaacctaaagaatacac cgtcggagta 900 agtaaaactg tcgttggact agatgaagat aagacgaaagacttcttgtt taatccaagt 960 gaaacgttgc aacaagagaa ttttccgctg agagatggtcagactaagga atttaaagta 1020 ccttatggaa cttctatatc aatagatgaa caagcctacgatgaatttaa agtatctgag 1080 tcaattacag aaaaaaatct agcaactggt gaagctgataaaacttatga tgctaccggc 1140 ttacaatccc tgacagtttc aggagacgta gatattagctttaccaatac acgtatcaag 1200 caaaaagtac gactacagaa agttaatgtc gaaaatgataataatttttt agcaggtgca 1260 gtttttgata tttatgaatc agatgctaat gggaataaagcttcacatcc tatgtattca 1320 gggctggtga caaacgataa aggcttgtta ttagtggatgctaataacta cctcagtttg 1380 ccagtaggaa aatactacct aacagagaca aaggcccctccagggtacct actacctaaa 1440 aatgatgata tatcagtatt agtgatttct acgggagttacctttgaaca aaatggtaat 1500 aatgcgacac caataaaaga gaatttagtg gatggaagtacagtatatac ttttaaaatt 1560 actaacagta aaggaacaga attgcctagt actggaggtattggaacaca catttatatc 1620 ctagttggtt tagctttagc tctaccatca ggattaatattatactatcg aaaaaaaata 1680 tga 1683 19 560 PRT Streptococcus agalactiae19 Met Val Ile Val Phe Arg Ile Ile Gln Ile Leu Gln Gly Ile Ile Ser 1 510 15 Lys Ile Leu Gln Val His Ile Ile Ile Ser Met Ile His Glu Ile Lys 2025 30 Ile Pro Thr Gln Leu Lys Met Pro Ile Ile Arg Gln Ile Leu Val Ser 3540 45 Ser Asn Val Asp Thr Thr Thr Lys Tyr Lys Tyr Val Lys Asp Ala Tyr 5055 60 Lys Leu Val Gly Trp Tyr Tyr Val Asn Pro Tyr Gly Ser Ile Arg Pro 6570 75 80 Tyr Asn Phe Ser Gly Ala Val Thr Gln Asp Ile Asn Leu Arg Ala Ile85 90 95 Trp Arg Lys Ala Gly Asp Tyr His Ile Ile Tyr Ser Asn Asp Ala Val100 105 110 Gly Thr Asp Gly Lys Pro Ala Leu Asp Ala Ser Gly Gln Gln LeuGln 115 120 125 Thr Ser Asn Glu Pro Thr Asp Pro Asp Ser Tyr Asp Asp GlySer His 130 135 140 Ser Ala Leu Leu Arg Arg Pro Thr Met Pro Asp Gly TyrArg Phe Arg 145 150 155 160 Gly Trp Trp Tyr Asn Gly Lys Ile Tyr Asn ProTyr Asp Ser Ile Asp 165 170 175 Ile Asp Ala His Leu Ala Asp Ala Asn LysAsn Ile Thr Ile Lys Pro 180 185 190 Val Ile Ile Pro Val Gly Asp Ile LysLeu Glu Asp Thr Ser Ile Lys 195 200 205 Tyr Asn Gly Asn Gly Gly Thr ArgVal Glu Asn Gly Asn Val Val Thr 210 215 220 Gln Val Glu Thr Pro Arg MetGlu Leu Asn Ser Thr Thr Thr Ile Pro 225 230 235 240 Glu Asn Gln Tyr PheThr Arg Thr Gly Tyr Asn Leu Ile Gly Trp His 245 250 255 His Asp Lys AspLeu Ala Asp Thr Gly Arg Val Glu Phe Thr Ala Gly 260 265 270 Gln Ser IleGly Ile Asp Asn Asn Leu Asp Ala Thr Asn Thr Leu Tyr 275 280 285 Ala ValTrp Gln Pro Lys Glu Tyr Thr Val Gly Val Ser Lys Thr Val 290 295 300 ValGly Leu Asp Glu Asp Lys Thr Lys Asp Phe Leu Phe Asn Pro Ser 305 310 315320 Glu Thr Leu Gln Gln Glu Asn Phe Pro Leu Arg Asp Gly Gln Thr Lys 325330 335 Glu Phe Lys Val Pro Tyr Gly Thr Ser Ile Ser Ile Asp Glu Gln Ala340 345 350 Tyr Asp Glu Phe Lys Val Ser Glu Ser Ile Thr Glu Lys Asn LeuAla 355 360 365 Thr Gly Glu Ala Asp Lys Thr Tyr Asp Ala Thr Gly Leu GlnSer Leu 370 375 380 Thr Val Ser Gly Asp Val Asp Ile Ser Phe Thr Asn ThrArg Ile Lys 385 390 395 400 Gln Lys Val Arg Leu Gln Lys Val Asn Val GluAsn Asp Asn Asn Phe 405 410 415 Leu Ala Gly Ala Val Phe Asp Ile Tyr GluSer Asp Ala Asn Gly Asn 420 425 430 Lys Ala Ser His Pro Met Tyr Ser GlyLeu Val Thr Asn Asp Lys Gly 435 440 445 Leu Leu Leu Val Asp Ala Asn AsnTyr Leu Ser Leu Pro Val Gly Lys 450 455 460 Tyr Tyr Leu Thr Glu Thr LysAla Pro Pro Gly Tyr Leu Leu Pro Lys 465 470 475 480 Asn Asp Asp Ile SerVal Leu Val Ile Ser Thr Gly Val Thr Phe Glu 485 490 495 Gln Asn Gly AsnAsn Ala Thr Pro Ile Lys Glu Asn Leu Val Asp Gly 500 505 510 Ser Thr ValTyr Thr Phe Lys Ile Thr Asn Ser Lys Gly Thr Glu Leu 515 520 525 Pro SerThr Gly Gly Ile Gly Thr His Ile Tyr Ile Leu Val Gly Leu 530 535 540 AlaLeu Ala Leu Pro Ser Gly Leu Ile Leu Tyr Tyr Arg Lys Lys Ile 545 550 555560 20 6 PRT Artificial sequence consensus 20 Leu Pro Ser Thr Gly Gly 15 21 6 PRT Artificial sequence consensus 21 Xaa Pro Xaa Thr Gly Gly 1 522 2714 DNA Streptococcus pneumoniae 22 caatcagaaa ttaccacgtg gcaatgttgactttatgaag gtggatggtc ggaccaatac 60 ctctcttcaa ggggcaatgt tcaaagtcatgaaagaagaa agcggacact atactcctgt 120 tcttcaaaat ggtaaggaag tagttgtaacatcagggaaa gatggtcgtt tccgagtgga 180 aggtctagag tatgggacat actatttatgggagctccaa gctccaactg gttatgttca 240 attaacatcg cctgtttcct ttacaatcgggaaagatact cgtaaggaac tggtaacagt 300 ggttaaaaat aacaagcgac cacggattgatgtgccagat acaggggaag aaaccttgta 360 tatcttgatg cttgttgcca ttttgttgtttggtagtggt tattatctta cgaaaaaacc 420 aaataactga tattcaatgt acatcattatgaaaaagata gcaggctgaa gggaagacca 480 gagtactctg aggtgatgtt aatcaggaatcatggtgatg tggcatgaat cacaataacg 540 gatatgaggc tgggcagatt gtgccagcctcattgtgggt tattgtttgt aaaacgatag 600 gactggtctg gtaatcattt taggaatggacaggactggg attctgattt aaaatggatg 660 gtgaatcaga aagaaatgag attttctcgtttctcttagc agataggatt gtctgttagg 720 aaaagcgata aaatgatgag tttgaagataaagggatgct gataaaaatg gtaaaaacaa 780 aaaagcaaaa acgaaataat ctcctattaggagtggtatt tttcattgga atggcggtaa 840 tggcgtatcc gctggtgtct cgcttgtattatcgagtgga atcaaatcaa caaattgctg 900 actttgataa ggaaaaagca acgttggatgaggctgacat tgatgaacga atgaaattgg 960 cacaagcctt caatgactct ttgaataatgtagtgagtgg cgatccttgg tcggaagaaa 1020 tgaagaaaaa agggcgagca gagtatgcacgtatgttaga aatccatgag cggatggggc 1080 atgtggaaat ccccgttatt gacgtggatttgccggttta tgctggtact gctgaagagg 1140 tattgcagca aggggctggg catctagagggaacttctct gccgatcgga ggcaattcga 1200 cccatgcggt gattacggca catacaggtttgccaacagc taagatgttt acggatttga 1260 ccaaacttaa agttggggat aagttttatgtgcacaatat caaggaagtg atggcctatc 1320 aagtggatca agtaaaggtg attgagccgacgaactttga tgatttattg attgtaccag 1380 gtcatgatta tgtgaccttg ctgacttgtacgccatacat gatcaatacc catcgtctat 1440 tggttcgggg gcatcggata ccgtacgtagcagaggttga ggaagaattt attgcagcaa 1500 acaaactcag tcatctctat cgctacctgttttatgtggc agttggtttg attgtgattc 1560 ttttatggat tattcgacgc ttgcgcaagaagaaaaaaca accggaaaag gctttgaagg 1620 cgctgaaagc agcaaggaag gaagtgaaggtggaggatgg acaacagtag acgttcacga 1680 aaaaaaggca caaaaaagaa gaaacatccgctgatccttc ttctgatttt cttagtagga 1740 ttcgccgttg cgatatatcc attggtgtctcgttattatt atcgtattga gtcaaacgag 1800 gttattaaag agtttgatga gacggtttcccagatggata aggcagaact tgaggagcgt 1860 tggcgcttgg ctcaagcctt caatgcgaccttgaaaccat ctgaaattct tgatcctttt 1920 acagagcaag agaaaaagaa aggcgtctcagaatatgcca atatgctaaa ggtccatgag 1980 cggattggct atgtggaaat tcctgcgattgatcaggaaa ttccgatgta tgtcggaacg 2040 agtgaggaca ttcttcagaa aggggcagggctgttagaag gggcttcgct gcctgttgga 2100 ggtgaaaata cccatacagt gatcactgctcacagaggat tgccaacggc agaattgttc 2160 agtcaattgg ataagatgaa aaaaggggatatcttttatc ttcacgtttt agatcaggtg 2220 ttggcctacc aagtggatca gatagtgacggtggagccga atgactttga gcctgtcttg 2280 attcaacatg gggaagatta tgcgaccttgttgacttgta caccgtatat gattaacagt 2340 catcgtctgt tggtacgtgg gaagcggattccgtatacgg caccaattgc agagcggaat 2400 cgagcggtga gagagcgtgg gcaattctggttgtggttat tactaggagc gatggcggtc 2460 atccttctct tgctgtatcg cgtgtatcgtaatcgacgga ttgtcaaagg actagaaaag 2520 caattggagg ggcgtcatgt caaggactaaactacgagcc ttattgggat acttgttgat 2580 gttggtagcc tgtttgattc ctatttattgttttggacag atggtgttgc agtctcttgg 2640 acaggtgaaa ggtcatgcta catttgtgaaatccatgaca actgaaatgt accaagaaca 2700 acagaaccat tctc 2714 23 297 PRTStreptococcus pneumoniae 23 Met Asp Asn Ser Arg Arg Ser Arg Lys Lys GlyThr Lys Lys Lys Lys 1 5 10 15 His Pro Leu Ile Leu Leu Leu Ile Phe LeuVal Gly Phe Ala Val Ala 20 25 30 Ile Tyr Pro Leu Val Ser Arg Tyr Tyr TyrArg Ile Glu Ser Asn Glu 35 40 45 Val Ile Lys Glu Phe Asp Glu Thr Val SerGln Met Asp Lys Ala Glu 50 55 60 Leu Glu Glu Arg Trp Arg Leu Ala Gln AlaPhe Asn Ala Thr Leu Lys 65 70 75 80 Pro Ser Glu Ile Leu Asp Pro Phe ThrGlu Gln Glu Lys Lys Lys Gly 85 90 95 Val Ser Glu Tyr Ala Asn Met Leu LysVal His Glu Arg Ile Gly Tyr 100 105 110 Val Glu Ile Pro Ala Ile Asp GlnGlu Ile Pro Met Tyr Val Gly Thr 115 120 125 Ser Glu Asp Ile Leu Gln LysGly Ala Gly Leu Leu Glu Gly Ala Ser 130 135 140 Leu Pro Val Gly Gly GluAsn Thr His Thr Val Ile Thr Ala His Arg 145 150 155 160 Gly Leu Pro ThrAla Glu Leu Phe Ser Gln Leu Asp Lys Met Lys Lys 165 170 175 Gly Asp IlePhe Tyr Leu His Val Leu Asp Gln Val Leu Ala Tyr Gln 180 185 190 Val AspGln Ile Val Thr Val Glu Pro Asn Asp Phe Glu Pro Val Leu 195 200 205 IleGln His Gly Glu Asp Tyr Ala Thr Leu Leu Thr Cys Thr Pro Tyr 210 215 220Met Ile Asn Ser His Arg Leu Leu Val Arg Gly Lys Arg Ile Pro Tyr 225 230235 240 Thr Ala Pro Ile Ala Glu Arg Asn Arg Ala Val Arg Glu Arg Gly Gln245 250 255 Phe Trp Leu Trp Leu Leu Leu Gly Ala Met Ala Val Ile Leu LeuLeu 260 265 270 Leu Tyr Arg Val Tyr Arg Asn Arg Arg Ile Val Lys Gly LeuGlu Lys 275 280 285 Gln Leu Glu Gly Arg His Val Lys Asp 290 295 24 894DNA Streptococcus pneumoniae 24 atggacaaca gtagacgttc acgaaaaaaaggcacaaaaa agaagaaaca tccgctgatc 60 cttcttctga ttttcttagt aggattcgccgttgcgatat atccattggt gtctcgttat 120 tattatcgta ttgagtcaaa cgaggttattaaagagtttg atgagacggt ttcccagatg 180 gataaggcag aacttgagga gcgttggcgcttggctcaag ccttcaatgc gaccttgaaa 240 ccatctgaaa ttcttgatcc ttttacagagcaagagaaaa agaaaggcgt ctcagaatat 300 gccaatatgc taaaggtcca tgagcggattggctatgtgg aaattcctgc gattgatcag 360 gaaattccga tgtatgtcgg aacgagtgaggacattcttc agaaaggggc agggctgtta 420 gaaggggctt cgctgcctgt tggaggtgaaaatacccata cagtgatcac tgctcacaga 480 ggattgccaa cggcagaatt gttcagtcaattggataaga tgaaaaaagg ggatatcttt 540 tatcttcacg ttttagatca ggtgttggcctaccaagtgg atcagatagt gacggtggag 600 ccgaatgact ttgagcctgt cttgattcaacatggggaag attatgcgac cttgttgact 660 tgtacaccgt atatgattaa cagtcatcgtctgttggtac gtgggaagcg gattccgtat 720 acggcaccaa ttgcagagcg gaatcgagcggtgagagagc gtgggcaatt ctggttgtgg 780 ttattactag gagcgatggc ggtcatccttctcttgctgt atcgcgtgta tcgtaatcga 840 cggattgtca aaggactaga aaagcaattggaggggcgtc atgtcaagga ctaa 894 25 3010 DNA Streptococcus pneumoniae 25tgttaggaaa agcgataaaa tgatgagttt gaagataaag ggatgctgat aaaaatggta 60aaaacaaaaa agcaaaaacg aaataatctc ctattaggag tggtattttt cattggaatg 120gcggtaatgg cgtatccgct ggtgtctcgc ttgtattatc gagtggaatc aaatcaacaa 180attgctgact ttgataagga aaaagcaacg ttggatgagg ctgacattga tgaacgaatg 240aaattggcac aagccttcaa tgactctttg aataatgtag tgagtggcga tccttggtcg 300gaagaaatga agaaaaaagg gcgagcagag tatgcacgta tgttagaaat ccatgagcgg 360atggggcatg tggaaatccc cgttattgac gtggatttgc cggtttatgc tggtactgct 420gaagaggtat tgcagcaagg ggctgggcat ctagagggaa cttctctgcc gatcggaggc 480aattcgaccc atgcggtgat tacggcacat acaggtttgc caacagctaa gatgtttacg 540gatttgacca aacttaaagt tggggataag ttttatgtgc acaatatcaa ggaagtgatg 600gcctatcaag tggatcaagt aaaggtgatt gagccgacga actttgatga tttattgatt 660gtaccaggtc atgattatgt gaccttgctg acttgtacgc catacatgat caatacccat 720cgtctattgg ttcgggggca tcggataccg tacgtagcag aggttgagga agaatttatt 780gcagcaaaca aactcagtca tctctatcgc tacctgtttt atgtggcagt tggtttgatt 840gtgattcttt tatggattat tcgacgcttg cgcaagaaga aaaaacaacc ggaaaaggct 900ttgaaggcgc tgaaagcagc aaggaaggaa gtgaaggtgg aggatggaca acagtagacg 960ttcacgaaaa aaaggcacaa aaaagaagaa acatccgctg atccttcttc tgattttctt 1020agtaggattc gccgttgcga tatatccatt ggtgtctcgt tattattatc gtattgagtc 1080aaacgaggtt attaaagagt ttgatgagac ggtttcccag atggataagg cagaacttga 1140ggagcgttgg cgcttggctc aagccttcaa tgcgaccttg aaaccatctg aaattcttga 1200tccttttaca gagcaagaga aaaagaaagg cgtctcagaa tatgccaata tgctaaaggt 1260ccatgagcgg attggctatg tggaaattcc tgcgattgat caggaaattc cgatgtatgt 1320cggaacgagt gaggacattc ttcagaaagg ggcagggctg ttagaagggg cttcgctgcc 1380tgttggaggt gaaaataccc atacagtgat cactgctcac agaggattgc caacggcaga 1440attgttcagt caattggata agatgaaaaa aggggatatc ttttatcttc acgttttaga 1500tcaggtgttg gcctaccaag tggatcagat agtgacggtg gagccgaatg actttgagcc 1560tgtcttgatt caacatgggg aagattatgc gaccttgttg acttgtacac cgtatatgat 1620taacagtcat cgtctgttgg tacgtgggaa gcggattccg tatacggcac caattgcaga 1680gcggaatcga gcggtgagag agcgtgggca attctggttg tggttattac taggagcgat 1740ggcggtcatc cttctcttgc tgtatcgcgt gtatcgtaat cgacggattg tcaaaggact 1800agaaaagcaa ttggaggggc gtcatgtcaa ggactaaact acgagcctta ttgggatact 1860tgttgatgtt ggtagcctgt ttgattccta tttattgttt tggacagatg gtgttgcagt 1920ctcttggaca ggtgaaaggt catgctacat ttgtgaaatc catgacaact gaaatgtacc 1980aagaacaaca gaaccattct ctcgcctaca atcaacgctt ggcttcgcaa aatcgcattg 2040tagatccttt tttggcggag ggatatgagg tcaattacca agtgtctgac gaccctgatg 2100cagtctatgg ttacttgtct attccaagtt tggaaatcat ggagccggtt tatttgggag 2160cagattatca tcatttaggg atgggcttgg ctcatgtgga tggtacaccg ctgcctctgg 2220atggtacagg gattcgctca gtgattgctg ggcaccgtgc agagccaagc catgtctttt 2280tccgccattt ggatcagcta aaagttggag atgctcttta ttatgataat ggccaggaaa 2340ttgtagaata tcagatgatg gacacagaga ttattttacc gtcggaatgg gaaaaattag 2400aatcggttag ctctaaaaat atcatgacct tgataacctg cgatccgatt cctaccttta 2460ataaacgctt attagtgaat tttgaacgag tcgctgttta tcaaaaatca gatccacaaa 2520cagctgcagt tgcgagggtt gcttttacga aagaaggaca atctgtatcg cgtgttgcaa 2580cctctcaatg gttgtaccgt gggctagtgg tactggcatt tctgggaatc ctgtttgttt 2640tgtggaagct agcacgttta ctacgaggga aataaaaaga aatgaaagga aagctaaggc 2700tgttcctttt tccggctctt tgtcaactgt agtgggttga aaaaaagcta agctcgagaa 2760aggacaaatt ttgtcctttc ttttttgata ttcagagcga taaaaatccg ttttttgaag 2820ttttcaaagt ttcgaaaacc aaaggcattg cgcttgataa gtttgatgag attattggtc 2880gcttccagtt tggcattaga atagtgtagt tgaagggcgt tgataacctt ttctttatct 2940ttgaggaagg ttttaaagac agtctgaaaa ataggatgaa cctgcttaag attgtcctcg 3000ataagttcga 3010 26 304 PRT Streptococcus pneumoniae 26 Met Leu Ile LysMet Val Lys Thr Lys Lys Gln Lys Arg Asn Asn Leu 1 5 10 15 Leu Leu GlyVal Val Phe Phe Ile Gly Met Ala Val Met Ala Tyr Pro 20 25 30 Leu Val SerArg Leu Tyr Tyr Arg Val Glu Ser Asn Gln Gln Ile Ala 35 40 45 Asp Phe AspLys Glu Lys Ala Thr Leu Asp Glu Ala Asp Ile Asp Glu 50 55 60 Arg Met LysLeu Ala Gln Ala Phe Asn Asp Ser Leu Asn Asn Val Val 65 70 75 80 Ser GlyAsp Pro Trp Ser Glu Glu Met Lys Lys Lys Gly Arg Ala Glu 85 90 95 Tyr AlaArg Met Leu Glu Ile His Glu Arg Met Gly His Val Glu Ile 100 105 110 ProVal Ile Asp Val Asp Leu Pro Val Tyr Ala Gly Thr Ala Glu Glu 115 120 125Val Leu Gln Gln Gly Ala Gly His Leu Glu Gly Thr Ser Leu Pro Ile 130 135140 Gly Gly Asn Ser Thr His Ala Val Ile Thr Ala His Thr Gly Leu Pro 145150 155 160 Thr Ala Lys Met Phe Thr Asp Leu Thr Lys Leu Lys Val Gly AspLys 165 170 175 Phe Tyr Val His Asn Ile Lys Glu Val Met Ala Tyr Gln ValAsp Gln 180 185 190 Val Lys Val Ile Glu Pro Thr Asn Phe Asp Asp Leu LeuIle Val Pro 195 200 205 Gly His Asp Tyr Val Thr Leu Leu Thr Cys Thr ProTyr Met Ile Asn 210 215 220 Thr His Arg Leu Leu Val Arg Glu His Arg IlePro Tyr Val Ala Glu 225 230 235 240 Val Glu Glu Glu Phe Ile Ala Ala AsnLys Leu Ser His Leu Tyr Arg 245 250 255 Tyr Leu Phe Tyr Val Ala Val GlyLeu Ile Val Ile Leu Leu Trp Ile 260 265 270 Ile Arg Arg Leu Arg Lys LysLys Lys Gln Pro Glu Lys Ala Leu Lys 275 280 285 Ala Leu Lys Ala Ala ArgLys Glu Val Lys Val Glu Asp Gly Gln Gln 290 295 300 27 915 DNAStreptococcus pneumoniae 27 atgctgataa aaatggtaaa aacaaaaaag caaaaacgaaataatctcct attaggagtg 60 gtatttttca ttggaatggc ggtaatggcg tatccgctggtgtctcgctt gtattatcga 120 gtggaatcaa atcaacaaat tgctgacttt gataaggaaaaagcaacgtt ggatgaggct 180 gacattgatg aacgaatgaa attggcacaa gccttcaatgactctttgaa taatgtagtg 240 agtggcgatc cttggtcgga agaaatgaag aaaaaagggcgagcagagta tgcacgtatg 300 ttagaaatcc atgagcggat ggggcatgtg gaaatccccgttattgacgt ggatttgccg 360 gtttatgctg gtactgctga agaggtattg cagcaaggggctgggcatct agagggaact 420 tctctgccga tcggaggcaa ttcgacccat gcggtgattacggcacatac aggtttgcca 480 acagctaaga tgtttacgga tttgaccaaa cttaaagttggggataagtt ttatgtgcac 540 aatatcaagg aagtgatggc ctatcaagtg gatcaagtaaaggtgattga gccgacgaac 600 tttgatgatt tattgattgt accaggtcat gattatgtgaccttgctgac ttgtacgcca 660 tacatgatca atacccatcg tctattggtt cgggggcatcggataccgta cgtagcagag 720 gttgaggaag aatttattgc agcaaacaaa ctcagtcatctctatcgcta cctgttttat 780 gtggcagttg gtttgattgt gattctttta tggattattcgacgcttgcg caagaagaaa 840 aaacaaccgg aaaaggcttt gaaggcgctg aaagcagcaaggaaggaagt gaaggtggag 900 gatggacaac agtag 915 28 2199 DNA Enterococcusfaecalis 28 actaaaattc gtttacttta tgcatttaaa tgaaaaagca gatcctacgaaaggctttaa 60 aaatgaggcg aatgttgata acggtcatac cgacgaccaa acaccaccaactgttgaagt 120 tgtgacaggt gggaaacgtt tcattaaagt cgatggcgat gtgacagcgacacaagcctt 180 ggcgggagct tcctttgtcg tccgtgatca aaacagcgac acagcaaattatttgaaaat 240 cgatgaaaca acgaaagcag caacttgggt gaaaacaaaa gctgaagcaactacttttac 300 aacaacggct gatggattag ttgatatcac agggcttaaa tacggtacctattatttaga 360 agaaactgta gctcctgatg attatgtctt gttaacaaat cggattgaatttgtggtcaa 420 tgaacaatca tatggcacaa cagaaaacct agtttcacca gaaaaagtaccaaacaaaca 480 caaaggtacc ttaccttcaa caggtggcaa aggaatctac gtttacttaggaagtggcgc 540 agtcttgcta cttattgcag gagtctactt tgctagacgt agaaaagaaaatgcttaatt 600 tctagcatca ccgaagaaat ttttagaaaa acaaagagcc tgggccaatcactgtcccag 660 gctctcatgc tttattttta aggaggaagc aatgaagtca aaaaagaaacgtcgtatcat 720 tgatggtttt atgattcttt tactgattat tggaataggt gcatttgcgtatccttttgt 780 tagcgatgca ttaaataact atctggatca acaaattatc gctcattatcaagcaaaagc 840 aagccaagaa aacaccaaag aaatggctga acttcaagaa aaaatggaaaagaaaaacca 900 agaattagcg aaaaaaggca gcaatcctgg attagatcct ttttctgaaacgcaaaaaac 960 aacgaaaaaa ccagacaaat cctattttga aagtcatacg attggtgttttaaccattcc 1020 aaaaataaat gtccgtttac caatttttga taaaacgaat gcattgctattggaaaaagg 1080 aagctccttg ttagaaggaa cctcctatcc tacaggtggt acgaatacacatgcggtcat 1140 ttcaggccat cgtggtctcc ctcaagccaa attatttaca gatttgccagaattaaaaaa 1200 aggcgatgaa ttttatatcg aagtcaatgg gaagacgctt gcttatcaagtagatcaaat 1260 aaaaaccgtt gaaccaactg atacaaaaga tttacacatt gagtctggccaagatctcgt 1320 cactttatta acttgcacac cgtatatgat aaacagtcat cggttattagttcgaggaca 1380 tcgtatccca tatcaaccag aaaaagcagc agcggggatg aaaaaagtggcacaacaaca 1440 aaatttacta ttatggacat tacttttaat tgcctgtgcg ttaattattagcggcttcat 1500 tatctggtac aagcgacgga aaaagacgac cagaaaacca aagtagtatgacgaaaaggc 1560 taaacatact aaaaaaaaga gtaaaaaaat agcttttcaa tttttaatcctccttatcgt 1620 gcataattga accagagaaa cagaagtatt aacgaaataa ctaaaagagcaagccctgaa 1680 taaaaagcga caaagggcca atcaatcgac tgtttaaatt cctgccaagtttggattttt 1740 ctgttttttt tcgcgctatc ctcaagcgtg agtaaataat tcaatagtaagaggagtagc 1800 aacaccgtga aatcatttgt ggtaaaaagc acatgtaaaa atagaatgacaaagacaaca 1860 cgggataaca ctcgattccg caaaattaaa aataacttag cacgcataataaaccaccat 1920 ttcttatcag agataatgaa tctgtttttg tctactcttt agttatatcataaaattctt 1980 aataatgaaa aaatgactcg agaaaataat tgaaaaaagt tttttttcctgaatcattat 2040 tttcgtaaat aaagaataaa cgtgttactc ttggcttatc aaatttggaaggagtgttaa 2100 aaatgaaata tctggatatt attgctttaa ttttattgat tgtcggaggtttaaactggt 2160 tattagttgg tgcatttaat tttgatttag ttgcaacaa 2199 29 284PRT Enterococcus faecalis 29 Met Lys Ser Lys Lys Lys Arg Arg Ile Ile AspGly Phe Met Ile Leu 1 5 10 15 Leu Leu Ile Ile Gly Ile Gly Ala Phe AlaTyr Pro Phe Val Ser Asp 20 25 30 Ala Leu Asn Asn Tyr Leu Asp Gln Gln IleIle Ala His Tyr Gln Ala 35 40 45 Lys Ala Ser Gln Glu Asn Thr Lys Glu MetAla Glu Leu Gln Glu Lys 50 55 60 Met Glu Lys Lys Asn Gln Glu Leu Ala LysLys Gly Ser Asn Pro Gly 65 70 75 80 Leu Asp Pro Phe Ser Glu Thr Gln LysThr Thr Lys Lys Pro Asp Lys 85 90 95 Ser Tyr Phe Glu Ser His Thr Ile GlyVal Leu Thr Ile Pro Lys Ile 100 105 110 Asn Val Arg Leu Pro Ile Phe AspLys Thr Asn Ala Leu Leu Leu Glu 115 120 125 Lys Gly Ser Ser Leu Leu GluGly Thr Ser Tyr Pro Thr Gly Gly Thr 130 135 140 Asn Thr His Ala Val IleSer Gly His Arg Gly Leu Pro Gln Ala Lys 145 150 155 160 Leu Phe Thr AspLeu Pro Glu Leu Lys Lys Gly Asp Glu Phe Tyr Ile 165 170 175 Glu Val AsnGly Lys Thr Leu Ala Tyr Gln Val Asp Gln Ile Lys Thr 180 185 190 Val GluPro Thr Asp Thr Lys Asp Leu His Ile Glu Ser Gly Gln Asp 195 200 205 LeuVal Thr Leu Leu Thr Cys Thr Pro Tyr Met Ile Asn Ser His Arg 210 215 220Leu Leu Val Arg Gly His Arg Ile Pro Tyr Gln Pro Glu Lys Ala Ala 225 230235 240 Ala Gly Met Lys Lys Val Ala Gln Gln Gln Asn Leu Leu Leu Trp Thr245 250 255 Leu Leu Leu Ile Ala Cys Ala Leu Ile Ile Ser Gly Phe Ile IleTrp 260 265 270 Tyr Lys Arg Arg Lys Lys Thr Thr Arg Lys Pro Lys 275 28030 855 DNA Enterococcus faecalis 30 atgaagtcaa aaaagaaacg tcgtatcattgatggtttta tgattctttt actgattatt 60 ggaataggtg catttgcgta tccttttgttagcgatgcat taaataacta tctggatcaa 120 caaattatcg ctcattatca agcaaaagcaagccaagaaa acaccaaaga aatggctgaa 180 cttcaagaaa aaatggaaaa gaaaaaccaagaattagcga aaaaaggcag caatcctgga 240 ttagatcctt tttctgaaac gcaaaaaacaacgaaaaaac cagacaaatc ctattttgaa 300 agtcatacga ttggtgtttt aaccattccaaaaataaatg tccgtttacc aatttttgat 360 aaaacgaatg cattgctatt ggaaaaaggaagctccttgt tagaaggaac ctcctatcct 420 acaggtggta cgaatacaca tgcggtcatttcaggccatc gtggtctccc tcaagccaaa 480 ttatttacag atttgccaga attaaaaaaaggcgatgaat tttatatcga agtcaatggg 540 aagacgcttg cttatcaagt agatcaaataaaaaccgttg aaccaactga tacaaaagat 600 ttacacattg agtctggcca agatctcgtcactttattaa cttgcacacc gtatatgata 660 aacagtcatc ggttattagt tcgaggacatcgtatcccat atcaaccaga aaaagcagca 720 gcggggatga aaaaagtggc acaacaacaaaatttactat tatggacatt acttttaatt 780 gcctgtgcgt taattattag cggcttcattatctggtaca agcgacggaa aaagacgacc 840 agaaaaccaa agtag 855 31 2687 DNACorynebacterium diphtheriae 31 gtggtccgga gtatgacaag aacgctccggttcaggtaaa cggcactggt aacggtaacg 60 atctcgtggt cacctctgac aagaacggcaacgtccactt cgagggcctg ttcgtctccg 120 acgaccagaa tgatccggga aagtcagctgcgcagcgctg ctacgtcctc gtcgagaccg 180 aggccccgac gggcttcgtt actccgaaagatgggacggt cttcccagtt gctgtaaaga 240 ttggacagac tgctaccact acctacgacgcaaaggtcga gaacgtcaag cgcgataccc 300 ctgacctgcc gctgaccggt ggcaagggtgtgctgttcct gatgattgcc ggtggtctgt 360 tgctgctggt tgctgttggt gctggtttcgtctttgtacg ccgtatcaac gagtaattga 420 tttgtcgcgt gattaaataa tcgcgttgcgccgcccaatg cagggcatca aatgccccgc 480 cggcgggcat aaacgccggc ggggtgcggtggctttccac cgcaccccca cattctttgt 540 cagagatttg ctgtttggcc tgtgccacccggcatccccc tatatgagaa acggacgtac 600 ctgtcatggt taccaccgcg tcaccgcgctctaccggacc ggataaccca gacgcgcaac 660 caaagcgtcg ttgggtcttt tccggactcgcattgtttgc gtgtataacg gcgctagccg 720 gcctcatgtt ggggttgtat ccatctactgcagcgtggtt taacgcccgc gaacaggcca 780 aactggtaga tctctatgat tccaaaattgaaaatgcaac ccctcttagc gcggaacaat 840 tacttgaact cgcgcaccgt tataacgaccgcctgaccgt aggcgctgct ctcgatccct 900 gggctaacgt cccccgcgga gcgggcaaagaagacggcga cggtatggcc tataaagacc 960 agttgcgtgt tgaccgtacg gatgtcatggctcgtatacg tatcccctct atcaaggtgg 1020 atctaccgat ctatcacggc acgagcgataacactctaaa gaagggcgct ggccatttgg 1080 aaggtacctc gttaccggtg ggaggaccacgcacccattc cgttatcact gcacaccgtg 1140 gcttagctga ggccaccatg ttcactaatctcaacaaggt tggggtaggg gatagattca 1200 ccattgaggt gatgggcgaa gtccttacgtatgaagtgcg tgaaactcgt gtggtcagcc 1260 cagaggacac taggttcctg caaactcaagacgatcgtga ccttgtcaca ctcgttactt 1320 gtactccgtt gggcatcaat acacatcgcattctggtgac agctgagcgc attactccca 1380 ccccgcaatc cgatatcgat gcagcacgtcaagcttccca aatcggcttc ccttggtggg 1440 cggtcatttt cgcagtggga tttagctttatcgccttgtt cttctggcgt tcgggttaca 1500 tgattcctcc aaagaagaag gaagaagacatcgaaagcga agctgatggc gatgaactct 1560 gaaacggcgg ggaaggaacc caacgtggtcagtaccgacg ctaaacactc caccggtacc 1620 agttccaatg cgggtaccgg tgagagctcagcgaaaaaga aagcgcagac ggcaattgct 1680 gcgatagtca tgcttttgtg cggactgttagggctggtga ttctgttcta tccagtcgtg 1740 tccactcaac ttaacaatta tgaacagtctaaactcgccc gacagtttgg tgcagacgct 1800 gcccaagctg accctgccgt agttgctgctgctcttgatg ctgcccatgc ctacaacgat 1860 tcgctagaaa atggacccct gcaggatccgtggaccggtg gagatagcac taaggatcct 1920 gcctatcagg catacgagaa actcttaggggaatatccgg cgatggctca gatctctatc 1980 ccggctattt ccgtgaacct tcccatttaccacgggacaa gcgacgccac actcctcaaa 2040 ggtgttgggc acctttacgg tactgcgctacccgttggtg gactggggac gcgttcggtt 2100 ctaacagcgc attcaggtat ccaaaaatcgaccttctttg acaatttaga aaaggtcaaa 2160 aagggtgacg ccatttatgt acgcaatattggtgagaccc tgaaatacca agtacgcgac 2220 atcgaaatca tccgtccagc ggagattgaccgtatccagc caatcccaga ccgagactta 2280 attaccctcg tgacctgtac accctatggaatcaataccc ataggctttt ggttactgcc 2340 gaacgtgtcc ctatggaacc cggtgaggcggaccgtgcat ttgccggtga cggaattgtc 2400 tggcagtggt ggatgaagct agctatcggtgtgttggtgg tcatccttct cctaactggg 2460 tggctcatta tccgtatttt gcgagctaggaaattcgcga agaaaacagc tggagcagac 2520 gctgctaaat ctgttgaacc tggtgatattgaggcgtcgc taagcgcttc agcggccgag 2580 gagtcccagt aatatgcaga aaccaatttccccaacacat gcaaacaccc aagcagtcgc 2640 ccattcctga aaggacgccc tactatgaagaagactcact tgttccg 2687 32 348 PRT Corynebacterium diphtheriae 32 MetAla Met Asn Ser Glu Thr Ala Gly Lys Glu Pro Asn Val Val Ser 1 5 10 15Thr Asp Ala Lys His Ser Thr Gly Thr Ser Ser Asn Ala Gly Thr Gly 20 25 30Glu Ser Ser Ala Lys Lys Lys Ala Gln Thr Ala Ile Ala Ala Ile Val 35 40 45Met Leu Leu Cys Gly Leu Leu Gly Leu Val Ile Leu Phe Tyr Pro Val 50 55 60Val Ser Thr Gln Leu Asn Asn Tyr Glu Gln Ser Lys Leu Ala Arg Gln 65 70 7580 Phe Gly Ala Asp Ala Ala Gln Ala Asp Pro Ala Val Val Ala Ala Ala 85 9095 Leu Asp Ala Ala His Ala Tyr Asn Asp Ser Leu Glu Asn Gly Pro Leu 100105 110 Gln Asp Pro Trp Thr Gly Gly Asp Ser Thr Lys Asp Pro Ala Tyr Gln115 120 125 Ala Tyr Glu Lys Leu Leu Gly Glu Tyr Pro Ala Met Ala Gln IleSer 130 135 140 Ile Pro Ala Ile Ser Val Asn Leu Pro Ile Tyr His Gly ThrSer Asp 145 150 155 160 Ala Thr Leu Leu Lys Gly Val Gly His Leu Tyr GlyThr Ala Leu Pro 165 170 175 Val Gly Gly Leu Gly Thr Arg Ser Val Leu ThrAla His Ser Gly Ile 180 185 190 Gln Lys Ser Thr Phe Phe Asp Asn Leu GluLys Val Lys Lys Gly Asp 195 200 205 Ala Ile Tyr Val Arg Asn Ile Gly GluThr Leu Lys Tyr Gln Val Arg 210 215 220 Asp Ile Glu Ile Ile Arg Pro AlaGlu Ile Asp Arg Ile Gln Pro Ile 225 230 235 240 Pro Asp Arg Asp Leu IleThr Leu Val Thr Cys Thr Pro Tyr Gly Ile 245 250 255 Asn Thr His Arg LeuLeu Val Thr Ala Glu Arg Val Pro Met Glu Pro 260 265 270 Gly Glu Ala AspArg Ala Phe Ala Gly Asp Gly Ile Val Trp Gln Trp 275 280 285 Trp Met LysLeu Ala Ile Gly Val Leu Val Val Ile Leu Leu Leu Thr 290 295 300 Gly TrpLeu Ile Ile Arg Ile Leu Arg Ala Arg Lys Phe Ala Lys Lys 305 310 315 320Thr Ala Gly Ala Asp Ala Ala Lys Ser Val Glu Pro Gly Asp Ile Glu 325 330335 Ala Ser Leu Ser Ala Ser Ala Ala Glu Glu Ser Gln 340 345 33 1047 DNACorynebacterium diphtheriae 33 atggcgatga actctgaaac ggcggggaaggaacccaacg tggtcagtac cgacgctaaa 60 cactccaccg gtaccagttc caatgcgggtaccggtgaga gctcagcgaa aaagaaagcg 120 cagacggcaa ttgctgcgat agtcatgcttttgtgcggac tgttagggct ggtgattctg 180 ttctatccag tcgtgtccac tcaacttaacaattatgaac agtctaaact cgcccgacag 240 tttggtgcag acgctgccca agctgaccctgccgtagttg ctgctgctct tgatgctgcc 300 catgcctaca acgattcgct agaaaatggacccctgcagg atccgtggac cggtggagat 360 agcactaagg atcctgccta tcaggcatacgagaaactct taggggaata tccggcgatg 420 gctcagatct ctatcccggc tatttccgtgaaccttccca tttaccacgg gacaagcgac 480 gccacactcc tcaaaggtgt tgggcacctttacggtactg cgctacccgt tggtggactg 540 gggacgcgtt cggttctaac agcgcattcaggtatccaaa aatcgacctt ctttgacaat 600 ttagaaaagg tcaaaaaggg tgacgccatttatgtacgca atattggtga gaccctgaaa 660 taccaagtac gcgacatcga aatcatccgtccagcggaga ttgaccgtat ccagccaatc 720 ccagaccgag acttaattac cctcgtgacctgtacaccct atggaatcaa tacccatagg 780 cttttggtta ctgccgaacg tgtccctatggaacccggtg aggcggaccg tgcatttgcc 840 ggtgacggaa ttgtctggca gtggtggatgaagctagcta tcggtgtgtt ggtggtcatc 900 cttctcctaa ctgggtggct cattatccgtattttgcgag ctaggaaatt cgcgaagaaa 960 acagctggag cagacgctgc taaatctgttgaacctggtg atattgaggc gtcgctaagc 1020 gcttcagcgg ccgaggagtc ccagtaa 104734 19 PRT Artificial sequence consensus/Streptococcus pyogenes 34 ThrLeu Leu Thr Cys Thr Pro Tyr Met Ile Asn Xaa His Arg Leu Leu 1 5 10 15Val Xaa Gly 35 19 PRT Corynebacterium diphtheriae 35 Thr Leu Val Thr CysThr Pro Tyr Gly Ile Asn Thr His Arg Leu Leu 1 5 10 15 Val Thr Ala 36 19PRT Streptococcus pyogenes 36 Thr Leu Val Thr Cys Thr Pro Tyr Gly ValAsn Thr Lys Arg Leu Leu 1 5 10 15 Val Arg Gly 37 150 PRT Streptococcuspyogenes 37 Ile Glu Asn Asn Asp Ile Met Gly Tyr Val Glu Val Pro Ser IleLys 1 5 10 15 Val Thr Leu Pro Ile Tyr His Tyr Thr Thr Asp Glu Val LeuThr Lys 20 25 30 Gly Ala Gly His Leu Phe Gly Ser Ala Leu Pro Val Gly GlyAsp Gly 35 40 45 Thr His Thr Val Ile Ser Ala His Arg Gly Leu Pro Ser AlaGlu Met 50 55 60 Phe Thr Asn Leu Asn Leu Val Lys Lys Gly Asp Thr Phe TyrPhe Arg 65 70 75 80 Val Leu Asn Lys Val Leu Ala Tyr Lys Val Asp Gln IleLeu Thr Val 85 90 95 Glu Pro Asp Gln Val Thr Ser Leu Ser Gly Val Met GlyLys Asp Tyr 100 105 110 Ala Thr Leu Val Thr Cys Thr Pro Tyr Gly Val AsnThr Lys Arg Leu 115 120 125 Leu Val Arg Gly His Arg Ile Ala Tyr His TyrLys Lys Tyr Gln Gln 130 135 140 Ala Lys Lys Ala Met Lys 145 150

What is claimed is:
 1. An isolated streptococcal polypeptide EmaA. 2.The EmaA polypeptide of claim 1 which comprises the amino acid sequenceset out in SEQ ID NO: 2, and analogs, variants and immunogenic fragmentsthereof
 3. An isolated streptococcal polypeptide EmaB.
 4. The EmaCpolypeptide of claim 3 which comprises the amino acid sequence set outin SEQ ID NO: 4, and analogs, variants and immunogenic fragmentsthereof.
 5. An isolated streptococcal polypeptide EmaC.
 6. The EmaCpolypeptide of claim 5 which comprises the amino acid sequence set outin SEQ ID NO: 6, and analogs, variants and immunogenic fragmentsthereof.
 7. An isolated streptococcal polypeptide EmaD.
 8. The EmaDpolypeptide of claim 7 which comprises the amino acid sequence set outin SEQ ID NO: 8, and analogs, variants and immunogenic fragmentsthereof.
 9. An isolated streptococcal polypeptide EmaE.
 10. The EmaEpolypeptide of claim 9 which comprises the amino acid sequence set outin SEQ ID NO: 10, and analogs, variants and immunogenic fragmentsthereof.
 11. The streptococcal polypeptide of any of claims 1, 3, 5, 7or 9 labeled with a detectable label.
 12. A vaccine comprising one ormore streptococcal polypeptides selected from the group of EmaA, EmaB,EmaC, EmaD and EmaE, and a pharmaceutically acceptable adjuvant.
 13. Thevaccine of claim 12, further comprising an antigen selected from thegroup consisting of: a. the polypeptide Spb1 or an immunogenic fragmentthereof; b. the polypeptide Spb2 or an immunogenic fragment thereof; c.the polypeptide C protein alpha antigen or an immunogenic fragmentthereof; d. the polypeptide Rib or an immunogenic fragment thereof; e.the polypeptide Lmb or an immunogenic fragment thereof; f. thepolypeptide C5a-ase or an immunogenic fragment thereof; g. Group Bstreptococcal polysaccharides or oligosaccharides; and h. anycombination of one or more of the foregoing.
 14. An immunogeniccomposition comprising one of more streptococcal polypeptides selectedfrom the group of EmaA, EmaB, EmaC, EmaD and EmaE, and apharmaceutically acceptable adjuvant.
 15. The immunogenic composition ofclaim 14, further comprising an antigen selected from the groupconsisting of: a. the polypeptide Spb1 of an immunogenic fragmentthereof; b. the polypeptide Spb2 or an immunogenic fragment thereof; c.the polypeptide C protein alpha antigen or an immunogenic fragmentthereof; d. the polypeptide Rib or an immunogenic fragment thereof; e.the polypeptide Lmb or an immunogenic fragment thereof; f thepolypeptide C5a-ase or an immunogenic fragment thereof; g. Group Bstreptococcal polysaccharides or oligosaccharides; and h. anycombination of one or more of the foregoing.
 16. A pharmaceuticalcomposition comprising one or more streptococcal polypeptides selectedfrom the group of EmaA, EmaB, EmaC, EmaD and EmaE, and apharmaceutically acceptable carrier.
 17. The pharmaceutical compositionof claim 16, further comprising an active ingredient selected from thegroup consisting of: a. Spb1 or Spb2 polypeptide; b. C protein alphaantigen; c. Rib polypeptide; d. Lmb polypeptide; e. C5a-ase polypeptide;f a Group B streptococcal polysaccharide or oligosaccharide; and g. ananti-streptococcal vaccine.
 18. A purified antibody to a streptococcalpolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE.19. A monoclonal antibody to a streptococcal polypeptide selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE.
 20. An immortal cell linethat produces a monoclonal antibody according to claim
 19. 21. Theantibody of any of claims 19 or 20 labeled with a detectable label. 22.The antibody of claim 21 wherein the label is selected from the groupconsisting of an enzyme, a chemical which fluoresces, and a radioactiveelement.
 23. A pharmaceutical composition comprising one or moreantibodies to a streptococcal protein selected from the group of EmaA,EmaB, EmaC, EmaD and EmaE, and a pharmaceutically acceptable carrier.24. A pharmaceutical composition comprising a combination of at leasttwo antibodies to streptococcal proteins and a pharmaceuticallyacceptable carrier, wherein at least one antibody to a protein selectedfrom the group of EmaA, EmaB, EmaC, EmaD and EmaE, is combined with atleast one antibody to a protein selected from the group of Spb1 andSpb2, Rib, Lmb, C5a-ase and C protein alpha antigen.
 25. An isolatednucleic acid which encodes the streptococcal polypeptide of claim 1, ora fragment thereof
 26. The isolated nucleic acid of claim 25, whereinthe nucleic acid is selected from the group consisting of: a. the DNAsequence of SEQ ID NO: 1; b. DNA sequences that hybridize to thesequence of subpart (a) under moderate stringency hybridizationconditions; c. DNA sequences capable of encoding the amino acid sequenceencoded by the DNA sequences of (a) or (b); d. degenerate variantsthereof; e. alleles thereof; and f. hybridizable fragments thereof. 27.An isolated nucleic acid which encodes the streptococcal polypeptide ofclaim
 3. 28. The isolated nucleic acid of claim 27, wherein the nucleicacid is selected from the group consisting of: a. the DNA sequence ofSEQ ID NO: 3; b. DNA sequences that hybridize to the sequence of subpart(a) under moderate stringency hybridization conditions; c. DNA sequencescapable of encoding the amino acid sequence encoded by the DNA sequencesof (a) or (b); d. degenerate variants thereof; e. alleles thereof; andf. hybridizable fragments thereof
 29. An isolated nucleic acid whichencodes the streptococcal polypeptide of claim
 5. 30. The isolatednucleic acid of claim 29, wherein the nucleic acid is selected from thegroup consisting of: a. the DNA sequence of SEQ ID NO: 5; b. DNAsequences that hybridize to the sequence of subpart (a) under moderatestringency hybridization conditions; c. DNA sequences capable ofencoding the amino acid sequence encoded by the DNA sequences of (a) or(b); d. degenerate variants thereof; e. alleles thereof; and f.hybridizable fragments thereof
 31. An isolated nucleic acid whichencodes the streptococcal polypeptide of claim
 7. 32. The isolatednucleic acid of claim 31, wherein the nucleic acid is selected from thegroup consisting of: a. the DNA sequence of SEQ ID NO: 7; b. DNAsequences that hybridize to the sequence of subpart (a) under moderatestringency hybridization conditions; c. DNA sequences capable ofencoding the amino acid sequence encoded by the DNA sequences of (a) or(b); d. degenerate variants thereof; e. alleles thereof; and f.hybridizable fragments thereof.
 33. An isolated nucleic acid whichencodes the streptococcal polypeptide of claim
 9. 34. The isolatednucleic acid of claim 33, wherein the nucleic acid is selected from thegroup consisting of: a. the DNA sequence of SEQ ID NO: 9; b. DNAsequences that hybridize to the sequence of subpart (a) under moderatestringency hybridization conditions; c. DNA sequences capable ofencoding the amino acid sequence encoded by the DNA sequences of (a) or(b); d. degenerate variants thereof; e. alleles thereof; and f.hybridizable fragments thereof
 35. A vector which comprises the nucleicacid of any of claims 25, 27, 29, 31 or 33 and a promoter.
 36. Thevector of claim 35, wherein the promoter comprises a bacterial, yeast,insect or mammalian promoter.
 37. The vector of claim 35, wherein thevector is a plasmid, cosmid: yeast artificial chromosome (YAC),bacteriophage or eukaryotic viral DNA.
 38. A host vector system for theproduction of a polypeptide which comprises the vector of claim 35 in asuitable host cell.
 39. The host vector system of claim 38, wherein thesuitable host cell comprises a prokaryotic or eukaryotic cell.
 40. Thenucleic acid of any of claims 25, 27, 29, 31 or 33 which is arecombinant DNA molecule.
 41. The recombinant DNA molecule of claim 40,wherein the DNA molecule is operatively linked to an expression controlsequence.
 42. A unicellular host transformed with a recombinant DNAmolecule of claim
 40. 43. A nucleic acid vaccine comprising therecombinant DNA molecule of claim
 40. 44. A method for detecting thepresence of a streptococcal polypeptide selected from the group of EmaA,EmaB, EmaC, EmaD and EmaE, wherein the streptococcal polypeptide ismeasured by: a. contacting a sample in which the presence or activity ofa streptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE is suspected with an antibody to the said streptococcalpolypeptide under conditions that allow binding of the streptococcalpolypeptide to antibody to occur; and b. detecting whether binding hasoccurred between the streptococcal polypeptide from the sample and theantibody; wherein the detection of binding indicates the presence oractivity of the streptococcal polypeptide in the sample.
 45. A methodfor detecting the presence of a bacterium having a gene encoding astreptococcal polypeptide selected from the group of emaA, emaB, emaC,emaD and emaE, comprising: a. contacting a sample in which the presenceor activity of the bacterium is suspected with an oligonucleotide whichhybridizes to a streptococcal polypeptide gene selected from the groupof emaA, emaB, emaC, emaD and emaE, under conditions that allow specifichybridization of the oligonucleotide to the gene to occur; and b.detecting whether hybridization has occurred between the oligonucleotideand the gene; wherein the detection of hybridization indicates thatpresence or activity of the bacterium in the sample.
 46. A method forpreventing infection with a bacterium that expresses a streptococcal Emapolypeptide comprising administering an immunogenically effective doseof a vaccine of claim 12 to a subject.
 47. A method for preventinginfection with a bacterium that expresses a streptococcal Emapolypeptide comprising administering an immunogenically effective doseof the immunogenic composition of claim 14 to a subject.
 48. A methodfor treating infection with a bacterium that expresses a streptococcalEma polypeptide comprising administering a therapeutically effectivedose of a pharmaceutical composition of claim 16 to a subject.
 49. Amethod for treating infection with a bacterium that expresses astreptococcal Ema polypeptide comprising administering a therapeuticallyeffective dose of a pharmaceutical composition of claim 23 to a subject.50. A method of inducing an immune response in a subject which has beenexposed to or infected with a streptococcal bacterium comprisingadministering to the subject an amount of the pharmaceutical compositionof claim 16, thereby inducing an immune response.
 51. A method forpreventing infection by a streptococcal bacterium in a subjectcomprising administering to the subject an amount of a pharmaceuticalcomposition of claim 23 and a pharmaceutically acceptable carrier ordiluent, thereby preventing infection by a streptococcal bacterium. 52.An isolated streptococcal Ema polypeptide comprising the amino acidsequence set out in SEQ ID NO:23.
 53. An isolated nucleic acid whichencodes the streptococcal polypeptide of claim
 52. 54. The isolatednucleic acid of claim 53, wherein the nucleic acid is selected from thegroup consisting of: a. the DNA sequence of SEQ ID NO: 24; b. DNAsequences that hybridize to the sequence of subpart (a) under moderatestringency hybridization conditions; c. DNA sequences capable ofencoding the amino acid sequence encoded by the DNA sequences of (a) or(b); d. degenerate variants thereof; e. alleles thereof; and f.hybridizable fragments thereof.
 55. An isolated streptococcal Emapolypeptide comprising the amino acid sequence set out in SEQ ID NO:26.56. An isolated nucleic acid which encodes the streptococcal polypeptideof claim
 55. 57. The isolated nucleic acid of claim 56, wherein thenucleic acid is selected from the group consisting of: a. the DNAsequence of SEQ ID NO: 27; b. DNA sequences that hybridize to thesequence of subpart (a) under moderate stringency hybridizationconditions; c. DNA sequences capable of encoding the amino acid sequenceencoded by the DNA sequences of (a) or (b); d. degenerate variantsthereof; e. alleles thereof; and f. hybridizable fragments thereof. 58.An isolated streptococcal Ema polypeptide comprising the amino acidsequence set out in SEQ ID NO:37.
 59. An isolated nucleic acid whichencodes the streptococcal polypeptide of claim
 58. 60. An enterococcalEma polypeptide comprising the amino acid sequence set out in SEQ IDNO:29.
 61. An isolated nucleic acid which encodes the enterococcalpolypeptide of claim
 60. 62. The isolated nucleic acid of claim 61,wherein the nucleic acid is selected from the group consisting of: a.the DNA sequence ofSEQ ID NO: 30; b. DNA sequences that hybridize to thesequence of subpart (a) under moderate stringency hybridizationconditions; c. DNA sequences capable of encoding the amino acid sequenceencoded by the DNA sequences of (a) or (b); d. degenerate variantsthereof; e. alleles thereof; and f. hybridizable fragments thereof. 63.An isolated Corynebacterium Ema polypeptide comprising the amino acidsequence set out in SEQ ID NO:
 32. 64. An isolated nucleic acid whichencodes the Corynebacterium polypeptide of claim
 63. 65. The isolatednucleic acid of claim 64, wherein the nucleic acid is selected from thegroup consisting of: a. the DNA sequence of SEQ ID NO: 33; b. DNAsequences that hybridize to the sequence of subpart (a) under moderatestringency hybridization conditions; c. DNA sequences capable ofencoding the amino acid sequence encoded by the DNA sequences of (a) or(b); d. degenerate variants thereof; e. alleles thereof; and f.hybridizable fragments thereof.
 66. An isolated bacterial polypeptidecomprising the amino acid sequence TLLTCTPYMINS/THRLLVR/KG (SEQ ID NO:34), wherein the polypeptide is not isolated from Actinomyces.
 67. Anisolated streptococcal polypeptide comprising the amino acid sequenceTLLTCTPYMINS/THRLLVR/KG (SEQ ID NO: 34).
 68. An isolated bacterialpolypeptide comprising the amino acid sequence TLVTCTPYGINTHRLLVTA (SEQID NO: 35).
 69. An isolated bacterial polypeptide comprising the aminoacid sequence TLVTCTPYGVNTKRLLVRG (SEQ ID NO: 36).
 70. An isolatedstreptococcal polypeptide comprising the amino acid sequenceTLVTCTPYGVNTKRLLVRG (SEQ ID NO: 36).
 71. An isolated polypeptide havingthe amino acid sequence selected from the group of TLLTCTPYNS/THRLLVR/KG (SEQ ID NO: 34), TLVTCTPYGINTHRLLVTA (SEQ ID NO: 35), andTLVTCTPYGVNTKRLLVRG (SEQ ID NO: 36).